Next Article in Journal
Development of Orally Administered γ-Tocotrienol (GT3) Nanoemulsion for Radioprotection
Next Article in Special Issue
Regulation of G Protein-Coupled Receptors by Ubiquitination
Previous Article in Journal
Cannabidiol Modulates the Expression of Alzheimer’s Disease-Related Genes in Mesenchymal Stem Cells
Previous Article in Special Issue
Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 18
Issue 1
10.3390/ijms18010027
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Novel Structural Approaches to Study GPCR Regulation

by
Marco A. Alfonzo-Méndez
,
Rocío Alcántara-Hernández
and
J. Adolfo García-Sáinz
*
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México 04510, Mexico
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2017, 18(1), 27; https://doi.org/10.3390/ijms18010027
Submission received: 4 November 2016 / Revised: 15 December 2016 / Accepted: 21 December 2016 / Published: 23 December 2016
(This article belongs to the Collection G Protein-Coupled Receptor Signaling and Regulation)
Versions Notes

Abstract

:
Background: Upon natural agonist or pharmacological stimulation, G protein-coupled receptors (GPCRs) are subjected to posttranslational modifications, such as phosphorylation and ubiquitination. These posttranslational modifications allow protein–protein interactions that turn off and/or switch receptor signaling as well as trigger receptor internalization, recycling or degradation, among other responses. Characterization of these processes is essential to unravel the function and regulation of GPCR. Methods: In silico analysis and methods such as mass spectrometry have emerged as novel powerful tools. Both approaches have allowed proteomic studies to detect not only GPCR posttranslational modifications and receptor association with other signaling macromolecules but also to assess receptor conformational dynamics after ligand (agonist/antagonist) association. Results: this review aims to provide insights into some of these methodologies and to highlight how their use is enhancing our comprehension of GPCR function. We present an overview using data from different laboratories (including our own), particularly focusing on free fatty acid receptor 4 (FFA4) (previously known as GPR120) and α1A- and α1D-adrenergic receptors. From our perspective, these studies contribute to the understanding of GPCR regulation and will help to design better therapeutic agents.

1. Introduction

G protein-coupled receptors (GPCRs) are the largest family of membrane proteins, comprising as many as 3%–5% of the genes encoding proteins in sequenced genomes [1,2]. A hallmark of GPCRs is their ability to interact with a large variety of chemically diverse ligands. For this reason, GPCRs mediate key physiological processes, ranging from vision and olfaction to signaling in diverse organs and systems, such as the central nervous system, endocrine and immune networks, respiratory and digestive tracks, and many others. Thus, it is not an overstatement to say that this family of receptors participates in all of the major functions of vertebrates. This family of receptors is also involved in many human diseases, including heart failure, hypertension, diabetes, prostate cancer and bronchial asthma, to mention a few [3]. It is estimated that 30%–40% of drugs prescribed to treat these diseases target GPCRs [4] and, due to the large number of orphan receptors and tools for pharmacological screening, this number will probably increase [5]. It is currently estimated that approximately 800 human GPCR sequences exist, and they have been classified on the basis of their ligand-binding characteristics, signaling and sequence. Indeed, Fredriksson et al. [1] classified human GPCR sequences into five main families: Glutamate, Rhodopsin, Adhesion, Frizzled/taste2, and Secretin.
GPCRs are also denominated as seven transmembrane domain receptors due to their characteristic structure of an extracellular amino terminus, an intracellular carboxyl terminus and seven membrane-spanning segments connected by intra- and extracellular loops [6,7]. Seven transmembrane receptors usually transduce their signals by coupling to heterotrimeric G proteins that modulate the activity of enzymes (such as adenylyl cyclase or phospholipase C) and ion channels. These effectors increase or reduce the concentrations of second messengers (cyclic AMP, IP3, diacylglycerol, calcium, etc.) modulating enzymes, such as protein kinases, to further propagate signals within the cell. Furthermore, there is also evidence indicating that β-arrestins participate in GPCR signaling [4].
Upon activation, GPCRs undergo conformational changes that induce interactions between their intracellular domains and downstream signaling molecules, such as G protein subunits and β-arrestins [4,7,8]. GPCR posttranslational modifications (PTMs) include phosphorylation, palmitoylation, acetylation, glycosylation and ubiquitination, among others. Such covalent changes seem to play roles in the signaling and regulation of GPCRs [9,10,11,12]. GPCRs are frequently glycosylated at the amino terminus facing outside the cell, whereas the intracellular carboxyl tail is a substrate for phosphorylation, palmitoylation and ubiquitination. Among these PTMs, phosphorylation seems to be a key player in determining receptor desensitization/resensitization cycles [10], while ubiquitination is associated with lysosomal sorting and degradation [13,14].
The physiological or pathological outcome of GPCR-mediated signaling depends on the molecules with which the receptors interact. Actually, the PTMs mentioned above can induce tertiary and quaternary protein structural changes that regulate receptors association to other molecules and their function [15,16]. Therefore, to understand receptor signaling and regulation and to design rational GPCR-targeted drugs, it is necessary to characterize the conformational and structural receptor dynamics, as well as the specific GPCR interactome.
X-ray crystallography is the gold standard for investigating the structures of proteins and higher-order protein complexes at atomic resolution. After a gap from the first solved rhodopsin receptor crystal structure [17], 20 structures from the Rhodopsin family, two from the Secretin family, two from the Glutamate family and one Frizzled GPCR structure have been reported to date (see [18,19,20,21] and the references therein). The Nobel Prize in Chemistry was awarded to Robert Lefkowitz and Brian Kobilka in 2012 for their contribution to knowledge on the function and structure of β-adrenergic receptors [4,7].
Despite these great achievements, studies dealing with GPCR crystal structures remain challenging because it is very difficult to express and purify a sufficient quantity of any GPCR in an intact and functionally active form for direct experimental definition of its structural properties and binding interactions [22]. X-ray crystallography also has limitations, such as difficulties in monitoring protein dynamics [23]. Thus, there is only one crystal structure of a signaling complex available, i.e., the β2 adrenoceptor/Gs complex [24]. Although the crystal structure of β-arrestin-1 in complex with a phosphorylated V2 vasopressin receptor carboxyl-terminal peptide has already been reported [25], no crystal structure of a GPCR subjected to PTMs has been reported yet. This makes it hard to characterize the signaling structure/function mechanism of GPCRs. For these reasons, it seems necessary to complement studies on GPCR structures with post-translational modifications and associated molecules using other methods.
Mass spectrometry (MS) is useful not only for identifying protein sequences [25] but also for examining their structures after PTMs, folding and dynamics [26,27]. After important improvements on MS methodology, this technique has emerged as an important addition to X-ray crystallography and has been used in studies of protein structure and dynamics. Moreover, MS experiments require a small amount of sample, have no mass limits, allow rapid processing and can be used in high-throughput analysis [28,29,30]. The aim of this review is to present how MS-based studies are contributing to our knowledge on GPCR posttranslational modifications, associated proteins and receptor function/structure.

2. Posttranslational Modifications of G Protein-Coupled Receptors (GPCRs)

PTMs are a series of processes that can vary among cells, depending on the repertoire of proteins expressed, whose functional repercussion can also be affected by different cell stimuli and conditions, which affect protein abundance, trafficking, cell location and stability [31,32,33]. Currently, 469 different PTMs are reported in the UniProt database (http://www.uniprot.org/): 326 in eukaryotes, 250 in bacteria, 80 in archaea, and more than 100 in Homo sapiens. According to PhosphoSitePlus (http://www.phosphosite.org/) [34], protein phosphorylation is the most common PTM and has been detected in approximately 17,500 proteins of the human proteome. Frequent modifications include ubiquitination (approximately 8100 proteins), lysine acetylation (approximately 6700 proteins), lysine methylation (approximately 2400 proteins) and glycosylation (approximately 4500 proteins). The less frequently reported PTMs include succinylation, SUMOylation, citrullination, neddylation, disulfide bonding and lipidation [35]. Importantly, >95% of these data have been derived from MS-based proteome studies. Some advances on the most studied PTM, phosphorylation, with reference to a closely associated PTM (ubiquitination), are subsequently presented.

2.1. Phosphorylation

The phosphorylation state of a given protein is the result of the activity of two groups of enzymes: protein kinases and protein phosphatases. Protein kinases are phosphotransferases that transfer the γ phosphate group from ATP into serine, threonine or tyrosine amino acid residues, whereas phosphatases are hydrolases that release phosphate groups from those residues. Many GPCRs are subject to phosphorylation, and there is evidence suggesting that this PTM is associated with receptor desensitization and intracellular trafficking [9,32,36,37]. Furthermore, evidence also exists for phosphorylation-independent attenuation of signaling [38]. GPCR phosphorylation can take place at tyrosine residues (see for example [39,40]); however, serine/threonine phosphorylation is much more common and has been more extensively studied. This observation likely reflects that serine/threonine protein kinases are among the main modulators of these receptors. Currently, it is indicated that in homologous desensitization (agonist-dependent response-attenuation), G protein-coupled-receptor kinases (GRKs) are the major players. However, in heterologous desensitization (receptor activation-independent signaling-attenuation), second messenger-dependent protein kinases, such as protein kinase C and protein kinase A, and other protein kinases are the major players [10]. However, it seems to be an oversimplification to attribute all receptor phosphorylation that takes place during homologous desensitization to the action of GRKs; see, for example, the role of EGF transactivation in α1B-adrenoceptor phosphorylation induced by noradrenaline [41].
GPCR phosphorylation seems to be required for appropriate association with β-arrestins, and a phosphate sensor has been suggested in β-arrestin-1 on the basis of functional and crystallographic data [25]. It is interesting that multiple phosphorylation sites exist in most GPCRs and that they are mainly located at their carboxyl termini and third intracellular loops. Experimental evidence has shown that different phosphorylation patterns exist on GPCRs, depending on what ligand activates them and the cell in which they are expressed on; such “phosphorylation codes” can determine receptor function [31,32,37,42]. Different patterns of GPCR phosphorylation exist in a cell-specific manner also with different ligands (total agonist, partial agonist or inverse agonist), which can differentially regulate such phosphorylation patterns. Interestingly, this can lead to preferential signaling towards one action rather than others, a phenomenon known as biased stimulation [31,32,37,42]. The possibility that PTMs might determine the pharmacodynamic behavior of ligands is a current area of intense research.
As already indicated, GPCR phosphorylation at the carboxyl terminus and intracellular loops leads to β-arrestin association and receptor uncoupling from cognate G proteins. This represents a switch from G protein mediated- to β-arrestin-mediated-signaling. β-Arrestins modulate numerous pathways, primarily through recruitment of β-arrestin adaptor proteins [43]. These include the clathrin adaptor AP2, NSF (Nethylmaleimide-sensitive fusion protein, an ATPase involved in membrane fusion), ARF6 (ADP-ribosylation factor 6, a small G protein involved in vesicular traffic which participate in GPCR endocytosis) and also kinases of the mitogen activated protein kinase pathway [43].
Phosphorylation site prediction by in silico analysis is also a very useful tool. Advances in bioinformatics have allowed frequent confirmation of predictions by in cellulo experiments. The quantitative MS-based approach, with its high sensitivity and specificity, and mutational analysis are being increasingly applied in protein phosphorylation analysis, including phosphorylation sites in GPCRs, overcoming the limitations of conventional approaches, such as sequence motif analysis and site-directed mutagenesis [44,45]. Through tandem affinity purification and MS, the amino acid sequence and phosphorylation sites can to be determined simultaneously and unequivocally. Many GPCRs are known to be phosphoproteins. However, only in a limited amount of cases have agonist-induced and heterologous desensitization-associated GPCR phosphorylation been studied by MS in cellulo. This is rapidly changing since more groups are using this approach, and it is very likely that much more information will be available within a few years. A non-comprehensive list of GPCRs that have been studied using MS is presented in Table 1. In many cases, functional consequences have been suggested on the basis of a series of consistent evidences; in others, site-directed mutagenesis studies have been used to support such possibilities.

2.2. Ubiquitination

Ubiquitination is the second most frequently studied PTM in GPCRs. Ubiquitination of GPCRs and ubiquitination of adaptor proteins have been shown to regulate the GPCR endocytic pathway or GPCR trafficking [59,60]. Ubiquitin is a polypeptide of 76 amino acids residues (~8.5 kDa) that when attached to GPCRs promote receptor sorting into degradative pathways, typically on early to late endosomes or maturing vesicular bodies. Receptor proteolysis leads to a decrease in the total number of GPCRs available for signaling, a process known as “down-regulation”, which forms a part of long-term signaling attenuation (reviewed in [61], see also the references therein).
Ubiquitination is carried out by an enzymatic cascade involving the sequential activity of three ligases: E1, E2 and E3. Through their action, ubiquitin moieties are covalently and reversible attached to protein substrates, mainly on the ε-amino groups of internal lysines or, less frequently, on the free amino group at the amino-terminus of substrates [59]. E3 ubiquitin ligases have been shown to interact with GPCRs either directly through non-canonical WW-domain-mediated interactions or indirectly through interactions involving adaptor proteins. Many GPCRs seem to be degraded by metallo-proteinases present in lysosomes. However, before this take place, the ubiquitin moieties are removed by ubiquitin-specific peptidases (named USPs) ([61] and the references therein). Interestingly, removal of the GPCR ubiquitin moiety by peptidases is also involved in recycling GPCRs to the cell-surface for resensitization [61].
Ubiquitination of ~40 different GPCRs has been reported and among the best studied, is the β2-adrenoceptor. Agonist-activation of this receptor leads to very rapid phosphorylation, followed by ubiquitination; this latter PTM decreases hours later, correlating with receptor degradation [62]. Using MS, the ubiquitinated sites have been mapped at K263 and K270, in the third intracellular loop, and at K348, K372 and K375, in the carboxyl terminus [63]. β2-Adrenoceptors, in which lysines were mutated, internalize into endosomes upon agonist-activation but are not degraded in the lysosomes [63,64]. In contrast, β2-adrenoceptors in which phosphorylation sites were mutated exhibited impaired ubiquitination as well as reduced β-arrestin interaction [62]. Therefore, agonist-stimulated β2-adrenoceptor ubiquitination requires prior receptor phosphorylation and β-arrestin binding [62,65]. Interestingly, carvedilol, a β-adrenergic “antagonist”, frequently used in medical practice for the treatment of cardiovascular diseases, induces β2-adrenoceptor-dependent β-arrestin signaling, receptor ubiquitination, internalization, endosomal trafficking, and degradation [65]. The action of carvedilol seems to take place through a different molecular processes [66]. The pharmacodynamic classification of drugs acting on GPCRs is becoming much more complex (i.e., agonist/antagonist, inverse agonists/biased agonist/alosteric modulators) [67,68,69] but also opening new paths for therapeutic intervention.
A similar case exists for fingolimod, an S1P1 receptor (agonist but functional antagonist) that induces rapid receptor phosphorylation, ubiquitination, internalization and degradation [70]. Down-regulation of S1P1 receptor seems to play a key role in lymphocyte migration [71] and astrocyte activation [72]. These actions seem to explain why this agent has found a therapeutic niche in the treatment of multiple sclerosis [73].
An excellent comprehensive review on ubiquitination/deubiquitination of GPCRs was recently published and readers are referred to this [65].

3. Mass Spectrometry

MS is becoming a key technique to identify and properly characterize protein PTMs, including phosphorylation, ubiquitination, glycosylation, and proteolytic cleavage, among others. This highly specialized technique allows researchers to obtain the amino acid sequences that are modified. With this key information, bioinformatic analysis can be performed to obtain predictions on the structural consequences of such modifications as well as the putative enzymes responsible, and experiments can be designed to test the PTM functional consequences and relevance of putative participants in these processes. Practically, MS is very important because it can allow critical information to be obtained within a reasonable time frame and with high sensitivity [74,75].
MS requires three basic steps: (1) sample preparation, which includes obtaining a GPCR sample in sufficient quantity (usually to be detectable in Coomassie blue-stained gels) and of reasonable purity, as well as its proteolysis products under carefully controlled conditions; (2) sample ionization and detection, which involves sample bombing with electrons, fragmentation and ion formation, separation of the components according to their mass-to-charge ratio and detection by the mass spectrometer; and (3) analysis of sample data.
Researchers working in the GPCR field, including our group, are frequently only marginally familiar with MS techniques. Although MS systems are becoming more common, as a part of the equipment present in academic facilities, the spectrometer’s sensitivity and the expertise of the scientist in charge of it remain critical. In our case, it has been essential to interact with scientists who specialize in MS in academic service units, while the use of qualified commercial services is also an option.
Like essentially all techniques, MS offers advantages and disadvantages. As indicated, its main advantage is the possibility of processing samples and obtaining reliable data within reasonable time frames. However, its disadvantages include its limited sensitivity to proteases and difficulty of sample ionization. The presence of contaminants in this high-sensitive assay can lead to misleading results. Simple MS is not quantitative, but quantitative data can be obtained through the use of heavy and light isotopes during cell culture, called isotope-coded affinity tag labeling [76]. In the case of GPCRs, the highly hydrophobic nature of their transmembrane domains remains a challenge. During the past decade, MS analysis has gained interest for elucidating protein–protein and ligand–protein interactions, validating interactions by double hybrid assays, co-immunoprecipitation assays and Western blot analysis. These different techniques together with advances in confocal microscopy, FRET (Förster resonance energy transfer)/BRET (Bioluminescence resonance energy transfer) and image analysis have greatly contributed to identification of cellular complexes and “interactomes”.

4. Tales of Three GPCRs

In the following subsections, we present some examples of what these new technologies have taught us. In the first case study, we present some recent advances on free fatty acid receptor 4 including its regulation by phosphorylation, identification and characterization of its phosphorylation sites, and their possible functional relevance. In the second case study, findings on proteins that associate and/or co-purify with α1A- and α1D-adrenoceptors and their possible relevance are presented. In these two subsections, the main emphasis is made on aspects that have already been published by different groups, and we are incorporating some findings from our laboratory that have not yet been disclosed and might be of interest to other groups working with these of other GPCRs.

4.1. Free Fatty Acid Receptor 4 (FFA4) Phosphorylation Sites

Free fatty acids are important metabolic fuels by themselves and are constituents of storage lipids, such as triglycerides and membrane lipids, including phospholipids and sphingolipids. In addition to these well-known biochemical roles, they exert functions as natural agonists for some nuclear receptors [77] and for a family of GPCRs, comprising four members: FFA1-FFA4, with differential affinity for distinct fatty acids [78]. FFA4 (previously known as GPR120) was deorphanized in 2005 [79], and it is abundantly expressed in the intestine, where it induces GLP-1 (glucagon-like peptide-1) release into the circulation, which modulates insulin secretion and participates in glucose homeostasis. Additional studies showed that FFA4-deficient mice develop obesity, glucose intolerance and a fatty liver. Furthermore, a dysfunctional variant of this receptor is associated with obesity and other metabolic disturbances in humans [80]. In addition, FFA4 activation induces insulin sensitization and anti-inflammatory effects [81], as well as a variety of other actions in different organs and tissues [78,82]. Our group and others have observed that FFA4 is a phosphoprotein whose phosphorylation state is modulated by agonists and activation of protein kinase C [42,83,84,85,86]. Unsurprisingly, agonist-induced phosphorylation does not seem to be mainly mediated by protein kinase C, but rather by other kinases, likely GRKs [83,85]. Agonist- and protein kinase C-mediated FFA4 phosphorylation seem to be associated with receptor internalization [83,86]. Our group was working on determining the phosphorylation sites using MS when a very elegant paper, employing MS, reported the FFA4 phosphorylation sites located at the carboxyl terminus and their importance, together with some acidic residues (E341, D348, and D355), in the receptor association with β-arrestin-2 (also known as arrestin-3) [42]. Using a mutagenesis approach, other group also reported some FFA4 phosphorylation sites located at the FFA4 carboxyl terminus [85]. In Table 2, the identified phosphorylated residues and the techniques employed are presented. Phosphorylation site predictions were obtained using the Group Based Prediction System (GPS algorithm 2.1 v) [87,88]. Further work has shown that these residues alter different aspects of receptor function [86]; thus, mutation to alanine of S357 and S361 (named cluster 2) markedly altered receptor internalization and arrestin 3 recruitment, whereas similar mutations of T347, T349 and S350 (cluster 1) had no effect on receptor internalization and had minor effect on arrestin 3 recruitment, but markedly altered FFA4-mediated Akt activation [86]. Interestingly, in a recent review of this receptor, it was shown that these phosphorylation sites are conserved in human, rat and mouse FFA4 orthologs [89].
It is important to mention that truncation of the full C-terminal tail did not restrict activation of heterotrimeric G proteins [42], which allows molecular distinction between G protein-mediated and arrestin-mediated signaling. This raises the possibility of ligand development for selective activation of one of these signaling pathways over the other. This is important considering the physiological roles of this receptor in metabolic syndrome, diabetes, adipose tissue development and inflammation [80,81,82,90]. We also show in Table 2 that there are putative phosphorylation sites not only in the carboxyl terminus but also in the third intracellular loop as predicted using the GPS algorithm [87,88] and also several suggested in a previous study [89]. MS data obtained by our group indicated that T242 is a phosphorylation site. At this point, the functional significance of this site remains to be determined (work in progress in our laboratory).

4.2. α1-Adrenoceptor Associated Proteins

α1-Adrenoceptors belong to a three-member (α1A, α1B, and α1D subtypes) subfamily of GPCRs that mediate the actions of adrenaline and noradrenaline [90]. This receptor subfamily participates in many physiological actions of catecholamines (regulation of blood pressure, urogenital functions, intermediary metabolism, among many others) and in the physiopathology of some diseases (hypertension, benign prostatic hyperplasia, among others) [91]. It is well-known that these adrenoceptors are phosphoproteins whose phosphorylation state is modulated by GRKs and protein kinase C and that their phosphorylation is associated with desensitization and internalization [9,92,93,94,95,96,97,98,99,100,101,102,103,104,105]. The elegant pioneering work of Cotecchia and coworkers identified, by site-directed mutagenesis, the GRK- and protein kinase C-target sites at the carboxyl terminus of α1B-adrenoceptors [99,100,101]. The phosphorylation sites in the other two α1-adrenoceptors have not yet been identified.
During our work on α1A-adrenoceptors using immunopurification and MS, we observed that an important number of proteins associate during the purification steps with this adrenoceptor, which were clearly and consistently observed in the different analyses performed. Some of these proteins are listed in Table 3. These include proteins involved in cell signaling, vesicular trafficking and degradation pathways, among others. It is clear that co-purification and detection by MS only suggests, but does not probe, direct or indirect (i.e., through the formation of mega-complexes or signalosomes) interactions with the GPCR of interest or play a role on signaling or regulation. However, published functional data are consistent with such possible interactions and might be provocative enough to be explored by different experimental approaches.
MS analysis suggested the possible interaction of α1A-adrenoceptors with enzymes and adaptor proteins previously reported as elements that participate in the regulation of this receptor subtype (phosphorylation/desensitization/internalization). This includes protein kinase C isoforms, such as protein kinase C α [106] and δ [107]. Surprisingly, the atypical isoform ζ was also detected. Other elements include subunits of phosphoinositide 3-kinase, which is known to participate in α1A-adrenoceptor phosphorylation and heterologous desensitization induced by okadaic acid and phorbol esters, as well as to co-immunoprecipitate with the adrenoceptor. Additionally, the regulatory subunit δ isoform of PP2A was also observed via MS of immuno-purified α1A-adrenoceptors.
Proteins, such as clathrin, dynamin, and Rabs, that participate in the internalization and trafficking of GPCRs have been identified as common members of GPCRs-complexes. In our MS studies, clathrin heavy chain, dynamin 2 and dynamin like-protein 1 were observed to be proteins that co-purify with α1A-adrenoceptors. It has be demonstrated that α1A-adrenoceptors co-localize with nuclear membrane protein lamina-associated protein 2 in adult cardiac myocytes, and it has been suggested to activate signaling at the nucleus [108] in our MS studies the presence of lamina 1 and lamina 2, exportins, and importins (Table 3).
Others proteins that were identified were members of the mitogen activated protein kinase family; myosin light-chain kinase; cytoskeletal proteins, such as actin, tubulin and myosin; and diverse elements of the degradation machinery, such as ubiquitin-protein ligase E3 and SUMO-activating enzyme subunit 2, among others (Table 3).
Differences in agonist-induced α1A-adrenoceptor phosphorylation, desensitization and internalization have been observed when the action of phenylethylamine agonists (such as phenylephrine and noradrenaline) were compared to imidazolines (such as oxymetazoline) [106]. The low-efficacy agonist, oxymetazoline, induces G protein-coupled receptor kinase-dependent α1A-adrenoceptor phosphorylation and it is followed by rapid desensitization and receptor internalization [106]. In contrast, phosphorylation of these receptors in response to noradrenaline largely depends on protein kinase C activity, it is not followed by clear desensitization, and the receptors undergo delayed internalization [106]. Loss of response after drug exposure is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline. α1A-Adrenoceptor activation seems to play a key role in development of benign prostatic hypertrophy and treatment with selective antagonists seem to be of great help, ameliorating urinary symptoms [109]. Antagonists that could induce receptor internalization/down-regulation could be of potential therapeutic use. Understanding the receptor’s PTM that take place under the action of different agents, and their cellular consequences might be of help in developing of more effective drugs with fewer undesirable effects.
α1D-Adrenoceptors play a major role in the control of blood pressure and in the pathogenesis of hypertension [110,111,112,113]. It has been observed that this receptor subtype exhibits intrinsic activity of functional importance [96,114,115,116,117,118]. This subtype has been particularly difficult to study. When expressed, it exhibits a predominant intracellular location [119], which seems be due to a domain located at the amino terminus [120,121,122]; therefore, amino terminus truncation is a suitable experimental procedure to achieve α1D-adrenoceptor expression at the plasma membrane [96,114,120,121,122]. Interestingly, recent detailed work has shown, using receptor affinity purification and MS, that in multiple human cell lines, α1D-adrenoceptors are expressed both as the full-length form and also as an amino terminus-truncated protein [123]. A cleavage site was identified at the L910/V91 site, and it was suggested that the proteolytic processing of the amino terminus is a physiological mechanism to achieve membrane location of α1D-adrenoceptors with optimal functional properties [123].
Hague and coworkers [124,125] have shown that the α1D-adrenoceptor carboxyl terminus associates through a PDZ-interacting motif with syntrophins, which increases receptor expression and stability. The dystrophin proteins, syntrophin, dystrobrevin, and utrophin, were identified as α1D-adrenoceptor-interacting proteins [125]. MS analysis of purified α1D-adrenoceptors evidenced these and other associated proteins (Table 4). In our experiments using MS of immuno-purified α1D-adrenoceptors, a series of dynamin and many nuclear proteins were observed; however, in most cases, their possible roles in the receptor’s signaling and regulation are unknown (Table 4).

5. Perspectives

MS has emerged as a powerful complementary structural tool that complements information gained through X-ray crystallography and nuclear magnetic resonance, which both provide high resolution three-dimensional structural information for GPCRs and GPCR complexes. MS is a sensitive tool for identifying GPCR PTMs (phosphorylation and ubiquitination). It can be easily anticipated that the number of GPCRs whose PTMs will be determined in the very near future will markedly increase. These approaches together with the use of fluorescent confocal microscopy, FRET (Förster resonance energy transfer) and BRET (Bioluminescence resonance energy transfer) provide and will continue to provide information to gain a much deeper understanding of GPCR signaling and regulation.
Knowledge on the PTMs that affect specific receptor sites has already produced advances in our understanding of organ function. In a very elegant paper, Bradley and coworkers [126] reported a genetically engineered mouse expressing a G protein-biased M3-muscarinic acetylcholine mutant receptor, which allowed defining the role of receptor phosphorylation in bronchial smooth muscle contraction in health and in a model of asthma. G protein-dependent signaling and receptor phosphorylation-dependent signaling were mapped, which potentially predicts the outcome of biased agents [126]. Such findings and developments provide better approaches for drug design.
Ghrelin is a pleiotropic hormone secreted by the stomach that promotes food-seeking behaviors and a positive energy balance [127]. Studies on ghrelin receptors have also provided exciting insight on the functional relevance of GPCRs’ phosphorylation sites. Using MS the phosphorylation sites on the ghrelin receptor, GHSR1a, were defined as S362, S363 and T366; these residues are located at the carboxyl terminus and seem to be primarily responsible for β-arrestin binding [58]. Ghrelin receptor knockout animals did not show any clear change in body weight or energy consumption [128]. Interestingly, rats with a mutation deleting the distal part of the receptor’s carboxyl-terminal tail showed increased body weight and adiposity and reduced glucose tolerance [129]. Such mutation, that maintains GHSR cell surface abundance but alters its signaling properties, provided important insight into the role of the ghrelin receptor stressing the physiological role of the carboxyl terminus as a suppressor of ghrelin sensitivity [129].
Clearly, the previous two examples provide compelling evidence on the importance of carefully studying GPCR structure and function to better understand physiology and pathology, needed for developing tools for pharmacology and therapeutics.

Acknowledgments

Research in our laboratory is partially supported by Grants from CONACYT [253156 and Fronteras 882] and DGAPA-UNAM [200915]. The generous help and advice of Ross Tomaino, MS, Associate Director of the Taplin Mass Spectrometry Facility, Harvard Medical School is gratefully acknowledged.

Author Contributions

The initial draft of this review was jointly written by Marco A. Alfonzo-Méndez and Rocío Alcántara-Hernández, and it was reviewed and further organized by J. Adolfo García-Sáinz. Marco Antonio Alfonzo-Méndez is a student of the Programa de Doctorado en Ciencias Bioquímicas-UNAM and a recipient of a fellowship from the Consejo Nacional de Ciencias y Tecnología (CONACYT).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

GPCRsG protein coupled-receptors
PTMsPosttranslational modifications
MSMass spectrometry
GRKsG protein-coupled receptor kinases
FRETFörster resonance energy transfer
BRETBioluminescence resonance energy transfer

References

  1. Fredriksson, R.; Lagerstrom, M.C.; Lundin, L.G.; Schioth, H.B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 2003, 63, 1256–1272. [Google Scholar] [CrossRef] [PubMed]
  2. Fredriksson, R.; Schioth, H.B. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 2005, 67, 1414–1425. [Google Scholar] [CrossRef] [PubMed]
  3. Blumenthal, D.K.; Garrison, J.C. Pharmacodynamics: Molecular mechanisms of drug action. In Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 12th ed.; Brunton, L., Ed.; McGraw Hill: New York, NY, USA, 2011; pp. 47–72. [Google Scholar]
  4. Lefkowitz, R.J. A brief history of G-protein coupled receptors (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 2013, 52, 6366–6378. [Google Scholar] [CrossRef] [PubMed]
  5. Jacobson, K.A. New paradigms in GPCR drug discovery. Biochem. Pharmacol. 2015, 98, 541–555. [Google Scholar] [CrossRef] [PubMed]
  6. Mustafi, D.; Palczewski, K. Topology of class A G protein-coupled receptors: Insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol. Pharmacol. 2009, 75, 1–12. [Google Scholar] [CrossRef] [PubMed]
  7. Kobilka, B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 2013, 52, 6380–6388. [Google Scholar] [CrossRef] [PubMed]
  8. Thomsen, A.R.; Plouffe, B.; Cahill, T.J., 3rd; Shukla, A.K.; Tarrasch, J.T.; Dosey, A.M.; Kahsai, A.W.; Strachan, R.T.; Pani, B.; Mahoney, J.P.; et al. GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling. Cell 2016, 166, 907–919. [Google Scholar] [CrossRef] [PubMed]
  9. García-Sáinz, J.A.; Vázquez-Prado, J.; Medina, L.C. α1-Adrenoceptors: Function and phosphorylation. Eur. J. Pharmacol. 2000, 389, 1–12. [Google Scholar] [CrossRef]
  10. Vázquez-Prado, J.; Casas-González, P.; García-Sáinz, J.A. G protein-coupled receptor cross-talk: Pivotal roles of protein phosphorylation and protein–protein interactions. Cell Signal. 2003, 15, 549–557. [Google Scholar] [CrossRef]
  11. García-Sáinz, J.A.; Romero-Ávila, M.T.; Molina-Muñoz, T.; García-Pasquel, M.-J. G-protein-coupled receptor-receptor tyrosine kinase crosstalk. Regulation of receptor sensitivity and roles of autocrine feedback loops and signal integration. Curr. Signal Transduct. Ther. 2008, 3, 174–182. [Google Scholar] [CrossRef]
  12. Xin, F.; Radivojac, P. Post-translational modifications induce significant yet not extreme changes to protein structure. Bioinformatics 2012, 28, 2905–2913. [Google Scholar] [CrossRef] [PubMed]
  13. Dores, M.R.; Lin, H.; N, J.G.; Mendez, F.; Trejo, J. The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting. Mol. Biol. Cell 2015, 26, 4660–4673. [Google Scholar] [CrossRef] [PubMed]
  14. Dores, M.R.; Trejo, J. Ubiquitination of G protein-coupled receptors: Functional implications and drug discovery. Mol. Pharmacol. 2012, 82, 563–570. [Google Scholar] [CrossRef] [PubMed]
  15. Venkatakrishnan, A.J.; Deupi, X.; Lebon, G.; Tate, C.G.; Schertler, G.F.; Babu, M.M. Molecular signatures of G-protein-coupled receptors. Nature 2013, 494, 185–194. [Google Scholar] [CrossRef] [PubMed]
  16. Li, J.; Philip, J.L.; Xu, X.; Theccanat, T.; Abdur Razzaque, M.; Akhter, S.A. β-Arrestins regulate human cardiac fibroblast transformation and collagen synthesis in adverse ventricular remodeling. J. Mol. Cell. Cardiol. 2014, 76, 73–83. [Google Scholar] [CrossRef] [PubMed]
  17. Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C.A.; Motoshima, H.; Fox, B.A.; Le Trong, I.; Teller, D.C.; Okada, T.; Stenkamp, R.E.; et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 2000, 289, 739–745. [Google Scholar] [CrossRef] [PubMed]
  18. Rosa, M.; Bech-Serra, J.J.; Canals, F.; Zajac, J.M.; Talmont, F.; Arsequell, G.; Valencia, G. Optimized proteomic mass spectrometry characterization of recombinant human mu-opioid receptor functionally expressed in pichia pastoris cell lines. J. Proteome Res. 2015, 14, 3162–3173. [Google Scholar] [CrossRef] [PubMed]
  19. Bortolato, A.; Dore, A.S.; Hollenstein, K.; Tehan, B.G.; Mason, J.S.; Marshall, F.H. Structure of class B GPCRs: New horizons for drug discovery. Br. J. Pharmacol. 2014, 171, 3132–3145. [Google Scholar] [CrossRef] [PubMed]
  20. Hollenstein, K.; de Graaf, C.; Bortolato, A.; Wang, M.W.; Marshall, F.H.; Stevens, R.C. Insights into the structure of class B GPCRs. Trends Pharmacol. Sci. 2014, 35, 12–22. [Google Scholar] [CrossRef] [PubMed]
  21. Dore, A.S.; Okrasa, K.; Patel, J.C.; Serrano-Vega, M.; Bennett, K.; Cooke, R.M.; Errey, J.C.; Jazayeri, A.; Khan, S.; Tehan, B.; et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 2014, 511, 557–562. [Google Scholar] [CrossRef] [PubMed]
  22. McCusker, E.C.; Bane, S.E.; O’Malley, M.A.; Robinson, A.S. Heterologous GPCR expression: A bottleneck to obtaining crystal structures. Biotechnol. Prog. 2007, 23, 540–547. [Google Scholar] [CrossRef] [PubMed]
  23. Hassell, A.M.; An, G.; Bledsoe, R.K.; Bynum, J.M.; Carter, H.L., 3rd; Deng, S.J.; Gampe, R.T.; Grisard, T.E.; Madauss, K.P.; Nolte, R.T.; et al. Crystallization of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 2007, 63, 72–79. [Google Scholar] [CrossRef] [PubMed]
  24. Rasmussen, S.G.; DeVree, B.T.; Zou, Y.; Kruse, A.C.; Chung, K.Y.; Kobilka, T.S.; Thian, F.S.; Chae, P.S.; Pardon, E.; Calinski, D.; et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 2011, 477, 549–555. [Google Scholar] [CrossRef] [PubMed]
  25. Shukla, A.K.; Manglik, A.; Kruse, A.C.; Xiao, K.; Reis, R.I.; Tseng, W.C.; Staus, D.P.; Hilger, D.; Uysal, S.; Huang, L.Y.; et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 2013, 497, 137–141. [Google Scholar] [CrossRef] [PubMed]
  26. Na, S.; Jeong, J.; Park, H.; Lee, K.J.; Paek, E. Unrestrictive identification of multiple post-translational modifications from tandem mass spectrometry using an error-tolerant algorithm based on an extended sequence tag approach. Mol. Cell. Proteom. 2008, 7, 2452–2463. [Google Scholar] [CrossRef] [PubMed]
  27. Kaltashov, I.A.; Eyles, S.J. Studies of biomolecular conformations and conformational dynamics by mass spectrometry. Mass Spectrom. Rev. 2002, 21, 37–71. [Google Scholar] [CrossRef] [PubMed]
  28. Lee, J.J.; Park, Y.S.; Lee, K.J. Hydrogen-deuterium exchange mass spectrometry for determining protein structural changes in drug discovery. Arch. Pharm. Res. 2015, 38, 1737–1745. [Google Scholar] [CrossRef] [PubMed]
  29. Huang, R.Y.; Chen, G. Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry. Anal. Bioanal. Chem. 2014, 406, 6541–6558. [Google Scholar] [CrossRef] [PubMed]
  30. Wei, H.; Mo, J.; Tao, L.; Russell, R.J.; Tymiak, A.A.; Chen, G.; Iacob, R.E.; Engen, J.R. Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: Methodology and applications. Drug Discov. Today 2014, 19, 95–102. [Google Scholar] [CrossRef] [PubMed]
  31. Butcher, A.J.; Prihandoko, R.; Kong, K.C.; McWilliams, P.; Edwards, J.M.; Bottrill, A.; Mistry, S.; Tobin, A.B. Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code. J. Biol. Chem. 2011, 286, 11506–11518. [Google Scholar] [CrossRef] [PubMed]
  32. Tobin, A.B.; Butcher, A.J.; Kong, K.C. Location, location, location...site-specific GPCR phosphorylation offers a mechanism for cell-type-specific signalling. Trends Pharmacol. Sci. 2008, 29, 413–420. [Google Scholar] [CrossRef] [PubMed]
  33. Norskov-Lauritsen, L.; Brauner-Osborne, H. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors. Eur. J. Pharmacol. 2015, 763, 233–240. [Google Scholar] [CrossRef] [PubMed]
  34. Hornbeck, P.V.; Zhang, B.; Murray, B.; Kornhauser, J.M.; Latham, V.; Skrzypek, E. PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations. Nucleic Acids Res. 2015, 43, D512–D520. [Google Scholar] [CrossRef] [PubMed]
  35. Pagel, O.; Loroch, S.; Sickmann, A.; Zahedi, R.P. Current strategies and findings in clinically relevant post-translational modification-specific proteomics. Expert Rev. Proteom. 2015, 12, 235–253. [Google Scholar] [CrossRef] [PubMed]
  36. Maeda, T.; Imanishi, Y.; Palczewski, K. Rhodopsin phosphorylation: 30 years later. Prog. Retin. Eye Res. 2003, 22, 417–434. [Google Scholar] [CrossRef]
  37. Tobin, A.B. G-protein-coupled receptor phosphorylation: Where, when and by whom. Br. J. Pharmacol. 2008, 153, S167–S176. [Google Scholar] [CrossRef] [PubMed]
  38. Ferguson, S.S. Phosphorylation-independent attenuation of GPCR signalling. Trends Pharmacol. Sci. 2007, 28, 173–179. [Google Scholar] [CrossRef] [PubMed]
  39. Baltensperger, K.; Karoor, V.; Paul, H.; Ruoho, A.; Czech, M.P.; Malbon, C.C. The β-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase. J. Biol. Chem. 1996, 271, 1061–1064. [Google Scholar] [CrossRef] [PubMed]
  40. Gavi, S.; Shumay, E.; Wang, H.Y.; Malbon, C.C. G-protein-coupled receptors and tyrosine kinases: Crossroads in cell signaling and regulation. Trends Endocrinol. Metab. 2006, 17, 48–54. [Google Scholar] [CrossRef] [PubMed]
  41. Casas-González, P.; García-Sáinz, J.A. Role of epidermal growth factor receptor transactivation in α1B-adrenoceptor phosphorylation. Eur. J. Pharmacol. 2006, 542, 31–36. [Google Scholar] [CrossRef] [PubMed]
  42. Butcher, A.J.; Hudson, B.D.; Shimpukade, B.; Alvarez-Curto, E.; Prihandoko, R.; Ulven, T.; Milligan, G.; Tobin, A.B. Concomitant action of structural elements and receptor phosphorylation determine arrestin-3 interaction with the free fatty acid receptor FFA4. J. Biol. Chem. 2014, 289, 18451–18465. [Google Scholar] [CrossRef] [PubMed]
  43. Shenoy, S.K.; Lefkowitz, R.J. β-Arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol. Sci. 2011, 32, 521–533. [Google Scholar] [CrossRef] [PubMed]
  44. Mouledous, L.; Froment, C.; Burlet-Schiltz, O.; Schulz, S.; Mollereau, C. Phosphoproteomic analysis of the mouse brain mu-opioid (MOP) receptor. FEBS Lett. 2015, 589, 2401–2408. [Google Scholar] [CrossRef] [PubMed]
  45. Alfonso-Garrido, J.; Garcia-Calvo, E.; Luque-Garcia, J.L. Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry. Anal. Bioanal. Chem. 2015, 407, 4893–4905. [Google Scholar] [CrossRef] [PubMed]
  46. Ohguro, H.; van Hooser, J.P.; Milam, A.H.; Palczewski, K. Rhodopsin phosphorylation and dephosphorylation in vivo. J. Biol. Chem. 1995, 270, 14259–14262. [Google Scholar] [CrossRef] [PubMed]
  47. Lee, K.A.; Craven, K.B.; Niemi, G.A.; Hurley, J.B. Mass spectrometric analysis of the kinetics of in vivo rhodopsin phosphorylation. Protein Sci. 2002, 11, 862–874. [Google Scholar] [CrossRef] [PubMed]
  48. Doronin, S.; Lin, F.; Wang, H.Y.; Malbon, C.C. The full-length, cytoplasmic C-terminus of the β 2-adrenergic receptor expressed in E. coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo. Protein Expr. Purif. 2000, 20, 451–461. [Google Scholar] [PubMed]
  49. Nobles, K.N.; Xiao, K.; Ahn, S.; Shukla, A.K.; Lam, C.M.; Rajagopal, S.; Strachan, R.T.; Huang, T.Y.; Bressler, E.A.; Hara, M.R.; et al. Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin. Sci. Signal. 2011, 4, ra51. [Google Scholar] [CrossRef] [PubMed]
  50. Gao, S.; Malbon, C.; Wang, H.Y. Probing the stoichiometry of β2-adrenergic receptor phosphorylation by targeted mass spectrometry. J. Mol. Signal. 2014, 9, 3. [Google Scholar] [CrossRef] [PubMed]
  51. Wu, S.; Birnbaumer, M.; Guan, Z. Phosphorylation analysis of G protein-coupled receptor by mass spectrometry: Identification of a phosphorylation site in V2 vasopressin receptor. Anal. Chem. 2008, 80, 6034–6037. [Google Scholar] [CrossRef] [PubMed]
  52. Busillo, J.M.; Armando, S.; Sengupta, R.; Meucci, O.; Bouvier, M.; Benovic, J.L. Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling. J. Biol. Chem. 2010, 285, 7805–7817. [Google Scholar] [CrossRef] [PubMed]
  53. Jeong, J.; Park, Y.U.; Kim, D.K.; Lee, S.; Kwak, Y.; Lee, S.A.; Lee, H.; Suh, Y.H.; Gho, Y.S.; Hwang, D.; et al. Cdk5 phosphorylates dopamine D2 receptor and attenuates downstream signaling. PLoS ONE 2013, 8, e84482. [Google Scholar] [CrossRef] [PubMed]
  54. Chen, X.; Bai, B.; Tian, Y.; Du, H.; Chen, J. Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling. J. Biol. Chem. 2014, 289, 31173–31187. [Google Scholar] [CrossRef] [PubMed]
  55. Bray, L.; Froment, C.; Pardo, P.; Candotto, C.; Burlet-Schiltz, O.; Zajac, J.M.; Mollereau, C.; Mouledous, L. Identification and functional characterization of the phosphorylation sites of the neuropeptide FF2 receptor. J. Biol. Chem. 2014, 289, 33754–33766. [Google Scholar] [CrossRef] [PubMed]
  56. Chen, C.; Chiu, Y.T.; Wu, W.; Huang, P.; Mann, A.; Schulz, S.; Liu-Chen, L.Y. Determination of sites of U50,488H-promoted phosphorylation of the mouse kappa opioid receptor (KOPR): Disconnect between KOPR phosphorylation and internalization. Biochem. J. 2016, 473, 497–508. [Google Scholar] [CrossRef] [PubMed]
  57. Zindel, D.; Engel, S.; Bottrill, A.R.; Pin, J.P.; Prezeau, L.; Tobin, A.B.; Bunemann, M.; Krasel, C.; Butcher, A.J. Identification of key phosphorylation sites in PTH1R which determine arrestin3 binding and fine tune receptor signaling. Biochem. J. 2016, 473, 4173–4192. [Google Scholar] [CrossRef] [PubMed]
  58. Bouzo-Lorenzo, M.; Santo-Zas, I.; Lodeiro, M.; Nogueiras, R.; Casanueva, F.F.; Castro, M.; Pazos, Y.; Tobin, A.B.; Butcher, A.J.; Camina, J.P. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ss-arrestins. Sci. Rep. 2016, 6, 22495. [Google Scholar] [CrossRef] [PubMed]
  59. Kennedy, J.E.; Marchese, A. Regulation of GPCR Trafficking by Ubiquitin. Prog. Mol. Biol. Transl. Sci. 2015, 132, 15–38. [Google Scholar] [PubMed]
  60. Skieterska, K.; Rondou, P.; van Craenenbroeck, K. Dopamine D4 receptor ubiquitination. Biochem. Soc. Trans. 2016, 44, 601–605. [Google Scholar] [CrossRef] [PubMed]
  61. Cottrell, G.S. Roles of proteolysis in regulation of GPCR function. Br. J. Pharmacol. 2013, 168, 576–590. [Google Scholar] [CrossRef] [PubMed]
  62. Shenoy, S.K.; McDonald, P.H.; Kohout, T.A.; Lefkowitz, R.J. Regulation of receptor fate by ubiquitination of activated β2-adrenergic receptor and β-arrestin. Science 2001, 294, 1307–1313. [Google Scholar] [CrossRef] [PubMed]
  63. Xiao, K.; Shenoy, S.K. β2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains. J. Biol. Chem. 2011, 286, 12785–12795. [Google Scholar] [CrossRef] [PubMed]
  64. Sarker, S.; Xiao, K.; Shenoy, S.K. A tale of two sites: How ubiquitination of a G protein-coupled receptor is coupled to its lysosomal trafficking from distinct receptor domains. Commun. Integr. Biol. 2011, 4, 528–531. [Google Scholar] [CrossRef] [PubMed]
  65. Jean-Charles, P.Y.; Snyder, J.C.; Shenoy, S.K. Chapter One—Ubiquitination and deubiquitination of G protein-coupled receptors. Prog. Mol. Biol. Transl. Sci. 2016, 141, 1–55. [Google Scholar] [PubMed]
  66. Han, S.O.; Xiao, K.; Kim, J.; Wu, J.H.; Wisler, J.W.; Nakamura, N.; Freedman, N.J.; Shenoy, S.K. MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β2-adrenergic receptors. J. Cell Biol. 2012, 199, 817–830. [Google Scholar] [CrossRef] [PubMed]
  67. Kenakin, T. Quantifying biased β-arrestin signaling. Handb. Exp. Pharmacol. 2014, 219, 57–83. [Google Scholar] [PubMed]
  68. Kenakin, T. The effective application of biased signaling to new drug discovery. Mol. Pharmacol. 2015, 88, 1055–1061. [Google Scholar] [CrossRef] [PubMed]
  69. Kenakin, T.P. A Pharmacology Primer: Theory, Applications, and Methods, 3rd ed.; Elsevier Academic Press: Amsterdam, The Netherlands, 2009; p. 389. [Google Scholar]
  70. Oo, M.L.; Thangada, S.; Wu, M.T.; Liu, C.H.; Macdonald, T.L.; Lynch, K.R.; Lin, C.Y.; Hla, T. Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor. J. Biol. Chem. 2007, 282, 9082–9089. [Google Scholar] [CrossRef] [PubMed]
  71. Rivera, J.; Proia, R.L.; Olivera, A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat. Rev. Immunol. 2008, 8, 753–763. [Google Scholar] [CrossRef] [PubMed]
  72. Choi, J.W.; Gardell, S.E.; Herr, D.R.; Rivera, R.; Lee, C.W.; Noguchi, K.; Teo, S.T.; Yung, Y.C.; Lu, M.; Kennedy, G.; et al. FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation. Proc. Natl. Acad. Sci. USA 2011, 108, 751–756. [Google Scholar] [CrossRef] [PubMed]
  73. Brinkmann, V.; Billich, A.; Baumruker, T.; Heining, P.; Schmouder, R.; Francis, G.; Aradhye, S.; Burtin, P. Fingolimod (FTY720): Discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 2010, 9, 883–897. [Google Scholar] [CrossRef] [PubMed]
  74. Chung, K.Y.; Day, P.W.; Velez-Ruiz, G.; Sunahara, R.K.; Kobilka, B.K. Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach. PLoS ONE 2013, 8, e54942. [Google Scholar] [CrossRef] [PubMed]
  75. Daulat, A.M.; Maurice, P.; Froment, C.; Guillaume, J.L.; Broussard, C.; Monsarrat, B.; Delagrange, P.; Jockers, R. Purification and identification of G protein-coupled receptor protein complexes under native conditions. Mol. Cell. Proteom. 2007, 6, 835–844. [Google Scholar] [CrossRef] [PubMed]
  76. AbdAlla, S.; Lother, H.; el Massiery, A.; Quitterer, U. Increased AT1 receptor heterodimers in preeclampsia mediate enhanced angiotensin II responsiveness. Nat. Med. 2001, 7, 1003–1009. [Google Scholar] [CrossRef] [PubMed]
  77. Chawla, A.; Repa, J.J.; Evans, R.M.; Mangelsdorf, D.J. Nuclear receptors and lipid physiology: Opening the X-files. Science 2001, 294, 1866–1870. [Google Scholar] [CrossRef] [PubMed]
  78. Stoddart, L.A.; Smith, N.J.; Milligan, G. International Union of Pharmacology. LXXI. Free fatty acid receptors FFA1, -2, and -3: Pharmacology and pathophysiological functions. Pharmacol. Rev. 2008, 60, 405–417. [Google Scholar] [CrossRef] [PubMed]
  79. Hirasawa, A.; Tsumaya, K.; Awaji, T.; Katsuma, S.; Adachi, T.; Yamada, M.; Sugimoto, Y.; Miyazaki, S.; Tsujimoto, G. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat. Med. 2005, 11, 90–94. [Google Scholar] [CrossRef] [PubMed]
  80. Ichimura, A.; Hirasawa, A.; Poulain-Godefroy, O.; Bonnefond, A.; Hara, T.; Yengo, L.; Kimura, I.; Leloire, A.; Liu, N.; Iida, K.; et al. Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human. Nature 2012, 483, 350–354. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  81. Oh, D.Y.; Talukdar, S.; Bae, E.J.; Imamura, T.; Morinaga, H.; Fan, W.; Li, P.; Lu, W.J.; Watkins, S.M.; Olefsky, J.M. GPR120 is an ω-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell 2010, 142, 687–698. [Google Scholar] [CrossRef] [PubMed]
  82. Hara, T.; Kashihara, D.; Ichimura, A.; Kimura, I.; Tsujimoto, G.; Hirasawa, A. Role of free fatty acid receptors in the regulation of energy metabolism. Biochim. Biophys. Acta 2014, 1841, 1292–1300. [Google Scholar] [CrossRef] [PubMed]
  83. Sánchez-Reyes, O.B.; Romero-Ávila, M.T.; Castillo-Badillo, J.A.; Takei, Y.; Hirasawa, A.; Tsujimoto, G.; Villalobos-Molina, R.; García-Sáinz, J.A. Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation. Eur. J. Pharmacol. 2014, 723, 368–374. [Google Scholar] [CrossRef] [PubMed]
  84. Burns, R.N.; Moniri, N.H. Agonism with the ω-3 fatty acids α-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor. Biochem. Biophys. Res. Commun. 2010, 396, 1030–1035. [Google Scholar] [CrossRef] [PubMed]
  85. Burns, R.N.; Singh, M.; Senatorov, I.S.; Moniri, N.H. Mechanisms of homologous and heterologous phosphorylation of FFA receptor 4 (GPR120): GRK6 and PKC mediate phosphorylation of Thr347, Ser350, and Ser357 in the C-terminal tail. Biochem. Pharmacol. 2014, 87, 650–659. [Google Scholar] [CrossRef] [PubMed]
  86. Prihandoko, R.; Alvarez-Curto, E.; Hudson, B.D.; Butcher, A.J.; Ulven, T.; Miller, A.M.; Tobin, A.B.; Milligan, G. Distinct phosphorylation clusters determine the signaling outcome of free fatty acid receptor 4/G protein-coupled receptor 120. Mol. Pharmacol. 2016, 89, 505–520. [Google Scholar] [CrossRef] [PubMed]
  87. Xue, Y.; Ren, J.; Gao, X.; Jin, C.; Wen, L.; Yao, X. GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Mol. Cell. Proteom. 2008, 7, 1598–1608. [Google Scholar] [CrossRef] [PubMed]
  88. Blom, N.; Gammeltoft, S.; Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 1999, 294, 1351–1362. [Google Scholar] [CrossRef] [PubMed]
  89. Liu, H.D.; Wang, W.B.; Xu, Z.G.; Liu, C.H.; He, D.F.; Du, L.P.; Li, M.Y.; Yu, X.; Sun, J.P. FFA4 receptor (GPR120): A hot target for the development of anti-diabetic therapies. Eur. J. Pharmacol. 2015, 763, 160–168. [Google Scholar] [CrossRef] [PubMed]
  90. Hieble, J.P.; Bylund, D.B.; Clarke, D.E.; Eikenburg, D.C.; Langer, S.Z.; Lefkowitz, R.J.; Minneman, K.P.; Ruffolo, R.R., Jr. International union of pharmacology. X. recommendation for nomenclature of α 1-adrenoceptors: Consensus update. Pharmacol. Rev. 1995, 47, 267–270. [Google Scholar] [PubMed]
  91. García-Sáinz, J.A.; Vázquez-Prado, J.; Villalobos-Molina, R. α1-Adrenoceptors: Subtypes, signaling, and roles in health and disease. Arch. Med. Res. 1999, 30, 449–458. [Google Scholar] [CrossRef]
  92. Cabrera-Wrooman, A.; Romero-Ávila, M.T.; García-Sáinz, J.A. Roles of the α1A-adrenergic receptor carboxyl tail in protein kinase C-induced phosphorylation and desensitization. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2010, 382, 499–510. [Google Scholar] [CrossRef] [PubMed]
  93. García-Sáinz, J.A.; Rodríguez-Pérez, C.E.; Romero-Ávila, M.T. Human α-1D adrenoceptor phosphorylation and desensitization. Biochem. Pharmacol. 2004, 67, 1853–1858. [Google Scholar] [CrossRef] [PubMed]
  94. García-Sáinz, J.A.; Romero-Ávila, M.T.; Alcántara-Hernández, R. Mechanisms involved in α1B-adrenoceptor desensitization. IUBMB Life 2011, 63, 811–815. [Google Scholar] [CrossRef] [PubMed]
  95. García-Sáinz, J.A.; Vázquez-Cuevas, F.G.; Romero-Ávila, M.T. Phosphorylation and desensitization of α1D-adrenergic receptors. Biochem. J. 2001, 353, 603–610. [Google Scholar] [CrossRef] [PubMed]
  96. Rodríguez-Pérez, C.E.; Romero-Ávila, M.T.; Reyes-Cruz, G.; García-Sáinz, J.A. Signaling properties of human α1D-adrenoceptors lacking the carboxyl terminus: Intrinsic activity, agonist-mediated activation, and desensitization. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2009, 380, 99–107. [Google Scholar] [CrossRef] [PubMed]
  97. Vázquez-Prado, J.; García-Sáinz, J.A. Effect of phorbol myristate acetate on α1-adrenergic action in cells expressing recombinant α1-adrenoceptor subtypes. Mol. Pharmacol. 1996, 50, 17–22. [Google Scholar] [PubMed]
  98. Vázquez-Prado, J.; Medina, L.C.; García-Sáinz, J.A. Activation of endothelin ETA receptors induces phosphorylation of α1B-adrenoreceptors in Rat-1 fibroblasts. J. Biol. Chem. 1997, 272, 27330–27337. [Google Scholar] [CrossRef] [PubMed]
  99. Diviani, D.; Lattion, A.L.; Cotecchia, S. Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the α1B-adrenergic receptor. J. Biol. Chem. 1997, 272, 28712–28719. [Google Scholar] [CrossRef] [PubMed]
  100. Diviani, D.; Lattion, A.L.; Larbi, N.; Kunapuli, P.; Pronin, A.; Benovic, J.L.; Cotecchia, S. Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the α1B-adrenergic receptor. J. Biol. Chem. 1996, 271, 5049–5058. [Google Scholar] [PubMed]
  101. Lattion, A.L.; Diviani, D.; Cotecchia, S. Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the α 1B-adrenergic receptor. J. Biol. Chem. 1994, 269, 22887–22893. [Google Scholar] [PubMed]
  102. Stanasila, L.; Abuin, L.; Dey, J.; Cotecchia, S. Different internalization properties of the α1A- and α1B-adrenergic receptor subtypes: The potential role of receptor interaction with β-arrestins and AP50. Mol. Pharmacol. 2008, 74, 562–573. [Google Scholar] [CrossRef] [PubMed]
  103. Morris, D.P.; Price, R.R.; Smith, M.P.; Lei, B.; Schwinn, D.A. Cellular trafficking of human α1A-adrenergic receptors is continuous and primarily agonist-independent. Mol. Pharmacol. 2004, 66, 843–854. [Google Scholar] [CrossRef] [PubMed]
  104. Price, R.R.; Morris, D.P.; Biswas, G.; Smith, M.P.; Schwinn, D.A. Acute agonist-mediated desensitization of the human α1A-adrenergic receptor is primarily independent of carboxyl terminus regulation: Implications for regulation of α1AAR splice variants. J. Biol. Chem. 2002, 277, 9570–9579. [Google Scholar] [CrossRef] [PubMed]
  105. Schwinn, D.A.; Kwatra, M.M. Expression and regulation of α1-adrenergic receptors in human tissues. In Catecholamines. Bridging Basic Science with Clinical Medicine; Goldstein, D.S., Eisenhofer, G., McCarty, R., Eds.; Academic Press: San Diego, CA, USA, 1998; pp. 390–394. [Google Scholar]
  106. Akinaga, J.; Lima, V.; Kiguti, L.R.; Hebeler-Barbosa, F.; Alcántara-Hernández, R.; García-Sáinz, J.A.; Pupo, A.S. Differential phosphorylation, desensitization, and internalization of α1A-adrenoceptors activated by norepinephrine and oxymetazoline. Mol. Pharmacol. 2013, 83, 870–881. [Google Scholar] [CrossRef] [PubMed]
  107. Shi, T.; Papay, R.S.; Perez, D.M. α1a-Adrenergic receptor prevents cardiac ischemic damage through PKCδa/GLUT1/4-mediated glucose uptake. J. Recept. Signal Transduct. Res. 2016, 36, 261–270. [Google Scholar] [CrossRef] [PubMed]
  108. Wu, S.C.; O’Connell, T.D. Nuclear compartmentalization of α1-adrenergic receptor signaling in adult cardiac myocytes. J. Cardiovasc. Pharmacol. 2015, 65, 91–100. [Google Scholar] [CrossRef] [PubMed]
  109. Schulman, C.C.; Cortvriend, J.; Jonas, U.; Lock, T.M.; Vaage, S.; Speakman, M.J. Tamsulosin, the first prostate-selective α1A-adrenoceptor antagonist. Analysis of a multinational, multicentre, open-label study assessing the long-term efficacy and safety in patients with benign prostatic obstruction (symptomatic BPH). European Tamsulosin Study Group. Eur. Urol. 1996, 29, 145–154. [Google Scholar] [PubMed]
  110. Villalobos-Molina, R.; Ibarra, M. α1-Adrenoceptors mediating contraction in arteries of normotensive and spontaneously hypertensive rats are of the α1D or α1A subtypes. Eur. J. Pharmacol. 1996, 298, 257–263. [Google Scholar] [CrossRef]
  111. Villalobos-Molina, R.; Ibarra, M. Vascular α1D-adrenoceptors: Are they related to hypertension? Arch. Med. Res. 1999, 30, 347–352. [Google Scholar] [CrossRef]
  112. Villalobos-Molina, R.; López-Guerrero, J.J.; Ibarra, M. Functional evidence of α1D-adrenoceptors in the vasculature of young and adult spontaneously hypertensive rats. Br. J. Pharmacol. 1999, 126, 1534–1536. [Google Scholar] [CrossRef] [PubMed]
  113. Tanoue, A.; Nasa, Y.; Koshimizu, T.; Shinoura, H.; Oshikawa, S.; Kawai, T.; Sunada, S.; Takeo, S.; Tsujimoto, G. The α1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction. J. Clin. Investig. 2002, 109, 765–775. [Google Scholar] [CrossRef] [PubMed]
  114. García-Sáinz, J.A.; Romero-Ávila, M.T.; Medina, L.C. α1D-Adrenergic receptors constitutive activity and reduced expression at the plasma membrane. Methods Enzymol. 2010, 484, 109–125. [Google Scholar] [PubMed]
  115. García-Sáinz, J.A.; Torres-Padilla, M.E. Modulation of basal intracellular calcium by inverse agonists and phorbol myristate acetate in rat-1 fibroblasts stably expressing α1D-adrenoceptors. FEBS Lett. 1999, 443, 277–281. [Google Scholar] [CrossRef]
  116. Gisbert, R.; Noguera, M.A.; Ivorra, M.D.; D’Ocon, P. Functional evidence of a constitutively active population of α1D-adrenoceptors in rat aorta. J. Pharmacol. Exp. Ther. 2000, 295, 810–817. [Google Scholar] [PubMed]
  117. Noguera, M.A.; Ivorra, M.D.; D’Ocon, P. Functional evidence of inverse agonism in vascular smooth muscle. Br. J. Pharmacol. 1996, 119, 158–164. [Google Scholar] [CrossRef] [PubMed]
  118. Ziani, K.; Gisbert, R.; Noguera, M.A.; Ivorra, M.D.; D’Ocon, P. Modulatory role of a constitutively active population of α1D-adrenoceptors in conductance arteries. Am. J. Physiol. Heart Circ. Physiol. 2002, 282, H475–H481. [Google Scholar] [CrossRef] [PubMed]
  119. McCune, D.F.; Edelmann, S.E.; Olges, J.R.; Post, G.R.; Waldrop, B.A.; Waugh, D.J.; Perez, D.M.; Piascik, M.T. Regulation of the cellular localization and signaling properties of the α1B- and α1D-adrenoceptors by agonists and inverse agonists. Mol. Pharmacol. 2000, 57, 659–666. [Google Scholar] [PubMed]
  120. Hague, C.; Chen, Z.; Pupo, A.S.; Schulte, N.A.; Toews, M.L.; Minneman, K.P. The N Terminus of the Human α1D-Adrenergic Receptor Prevents Cell Surface Expression. J. Pharmacol. Exp. Ther. 2004, 309, 388–397. [Google Scholar] [CrossRef] [PubMed]
  121. Hague, C.; Lee, S.E.; Chen, Z.; Prinster, S.C.; Hall, R.A.; Minneman, K.P. Heterodimers of α1B- and α1D-adrenergic receptors form a single functional entity. Mol. Pharmacol. 2006, 69, 45–55. [Google Scholar] [PubMed]
  122. Hague, C.; Uberti, M.A.; Chen, Z.; Hall, R.A.; Minneman, K.P. Cell surface expression of α1d-adrenergic receptors is controlled by heterodimerization with α1b-adrenergic receptors. J. Biol. Chem. 2004, 279, 15541–15549. [Google Scholar] [CrossRef] [PubMed]
  123. Kountz, T.S.; Lee, K.S.; Aggarwal-Howarth, S.; Curran, E.; Park, J.M.; Harris, D.A.; Stewart, A.; Hendrickson, J.; Camp, N.D.; Wolf-Yadlin, A.; et al. Endogenous N-terminal domain cleavage modulates α1d-adrenergic receptor pharmacodynamics. J. Biol. Chem. 2016, 291, 18210–18221. [Google Scholar] [CrossRef] [PubMed]
  124. Chen, Z.; Hague, C.; Hall, R.A.; Minneman, K.P. Syntrophins regulate α1D-adrenergic receptors through a PDZ domain-mediated interaction. J. Biol. Chem. 2006, 281, 12414–12420. [Google Scholar] [CrossRef] [PubMed]
  125. Lyssand, J.S.; Defino, M.C.; Tang, X.B.; Hertz, A.L.; Feller, D.B.; Wacker, J.L.; Adams, M.E.; Hague, C. Blood pressure is regulated by an α1D-adrenergic receptor/dystrophin signalosome. J. Biol. Chem. 2008, 283, 18792–18800. [Google Scholar] [CrossRef] [PubMed]
  126. Bradley, S.J.; Wiegman, C.H.; Iglesias, M.M.; Kong, K.C.; Butcher, A.J.; Plouffe, B.; Goupil, E.; Bourgognon, J.M.; Macedo-Hatch, T.; LeGouill, C.; et al. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction. Proc. Natl. Acad. Sci. USA 2016, 113, 4524–4529. [Google Scholar] [CrossRef] [PubMed]
  127. Kojima, M.; Hosoda, H.; Date, Y.; Nakazato, M.; Matsuo, H.; Kangawa, K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999, 402, 656–660. [Google Scholar] [CrossRef] [PubMed]
  128. Zigman, J.M.; Nakano, Y.; Coppari, R.; Balthasar, N.; Marcus, J.N.; Lee, C.E.; Jones, J.E.; Deysher, A.E.; Waxman, A.R.; White, R.D.; et al. Mice lacking ghrelin receptors resist the development of diet-induced obesity. J. Clin Investig. 2005, 115, 3564–3572. [Google Scholar] [CrossRef] [PubMed]
  129. Chebani, Y.; Marion, C.; Zizzari, P.; Chettab, K.; Pastor, M.; Korostelev, M.; Geny, D.; Epelbaum, J.; Tolle, V.; Morisset-Lopez, S.; et al. Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite. Sci. Signal. 2016, 9, ra39. [Google Scholar] [CrossRef] [PubMed]
Table
Table 1. G protein-coupled receptor (GPCR) studied using mass spectrometry. Amino acids in red indicate phosphorylation sites. Domain (DOM), carboxyl-terminus (C-term), third intracellular loop (3IL), references (Ref.).
Table 1. G protein-coupled receptor (GPCR) studied using mass spectrometry. Amino acids in red indicate phosphorylation sites. Domain (DOM), carboxyl-terminus (C-term), third intracellular loop (3IL), references (Ref.).
GPCRMain Phospho-Peptides Identified by MSDOMFunctional RoleRef.
RhodopsinDDDAS334ATASKTEC-termInactivation[46]
DDDASATAS338KTEC-term
RhodopsinDDDAS334ATASKTEC-termInactivation[47]
DDDASATAS338KTEC-term
DDDASATASKTETS343QVAPAC-term
β2-AdrenergicLPGT384EDFVGHQGT393VPS396DNIDS401QGRNCS407T408NDC-termIn vitro phosphorylation. Desensitization[48]
LPGT384EDFVGHQGT393VPS396DNIDS401QGRNCS407T408NDC-term
S411LLDLPGT384EDFVGHQGT393VPS396DNIDS401QGRNCS407T408NDC-term
β2-AdrenergicFHVQNLS246QVEQDGRT3ILDesensitization[49,50]
RS261SKFCLKE3IL
RSS262KFCLKE3IL
AYGNYS355SNGNTGEQSGYHVEQEKC-term
AYGNYSS356NGNTGEQSGYHVEQEKC-term
LLCEDLPGTEDFVGHQGTVPSDNIDS401QGRC-term
V2-vasopressinTGS255PGEGAHVSAAVAK3ILNot suggested[51]
CXCR4ALTSVSRGS323S324LKILC-termDesensitization Internalization Signaling[52]
Muscarinic M3PS384SDNLQVPD3ILSignaling[31]
QAQKS412MDDR3IL
QS577VIFHKC-term
Dopamine 2HGLHSTPDS321PAKPEK3ILDesensitization Internalization[53]
GPR120/FFA4GAILT347DTS350VKRC-termDesensitization and Recruitment of arrestin 3[42]
GAILTDT349S350VKRC-term
RNDLS357IISGYPYDVPDYAC-term
Apelin (APJ)SAS345YSSGHSQGPGPNMGKC-termBiased signaling[54]
SASYSS348GHSQGPGPNMGKC-term
Neuropeptide FF2 (NPFF2)AKS369HVLINT375S376NQLVQESTFQNPHGETLLYRC-termDesensitization[55]
KS398AEKPQQELVMEELKC-term
ETTNSS418EIESAMVSKC-term
µ-OpioidEFCIPTSSTIEQQNS363ARC-termInternalization[44]
EHPS375TANTVDRC-term
QNT370REHPSTANTVDRC-term
κ-OpioidRQS356T357NRVRNTVQDPASMRDVGGMNKPVTHHHHHRC-termInternalization[56]
QSTNRVRNT363VQDPAS369MRDC-term
Parathyroid hormone receptor 1 (PTHR1)S473WSRWTLALDKRC-termInteraction with β-arrestins[57]
SGS491SSYSYGPMVSHTSVTNVGPRC-term
SGSS492S493YSYGPMVSHTSVTNVGPRC-term
SGSSSYSYGPMVSHT503S504VTNVGPRC-term
VGLGLPLS518PRC-term
PGTPALET548LETTPPAMAAPKC-term
PGTPALETLETT552PPAMAAPKC-term
Growth hormone secretagogue receptor (GHSR1)KLS349T/350LKDESSRC-termEndocytosis and recruitment of β-arrestins[58]
AWTES362SINT366C-term
AWTESS363INT366C-term
Table
Table 2. FFA4 receptor phosphorylation sites. Amino acids in red indicate phosphorylation sites; references (Ref.).
Table 2. FFA4 receptor phosphorylation sites. Amino acids in red indicate phosphorylation sites; references (Ref.).
FFA4 Receptor Carboxyl Tail SequenceTechniqueRef.
CRNEWKKIFCCFWFPEKGAILT347DT349S350VKRNDLS357IIS360GIn silico[87,88]
CRNEWKKIFCCFWFPEKGAILT347DTS350VKRNDLS357IISGMutagenesis[85]
CRNEWKKIFCCFWFPEKGAILT347DT349S350VKRNDLS357II SGMS[42]
CRNEWKKIFCCFWFPEKGAILT347DT349S350VKRNDLS357IIS360GMS[86]
CRNEWKKIFCCFWFPEKGAILTDTS350VKRNDLS357IIS360GMSOur data
FFA4 Receptor Intracellular Loop 3 SequenceTechniqueRef.
S226YS228KILQITKAS237RKRLT242VSLAYSES250HQIRVS256QQDFRLFRT265LFLIn silico GPS[87,88]
SYSKILQITKAS237RKRLT242VSLAYSESHQIRVS256QQDFRLFRTLFLIn silico[89]
SYSKILQITKASRKRLT242VSLAYSEHQIRVSQQDFRLFRTLFLMSOur data
Table
Table 3. Protein detected MS of immunopurified α1A-adrenoceptors. PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PP2A, protein phosphatase 2A; STAT, signal transducer and activator of transcription.
Table 3. Protein detected MS of immunopurified α1A-adrenoceptors. PKC, protein kinase C; PI3K, phosphoinositide 3-kinase; PP2A, protein phosphatase 2A; STAT, signal transducer and activator of transcription.
Detected ProteinFunctionPossible Role
PKC α, δ and ζSerine/threonine protein kinaseDesensitization
PI3KPhosphoinositide-dependent protein kinaseDesensitization
PP2ASerine/threonine protein phosphataseResensitization
Dynamin 2Scission of newly formed vesicles from de plasma membraneInternalization
ClathrinFormation of coated vesiclesInternalization
STAT1 and 3Signal transducer and activator of transcriptionUnknown
MAD2 and 4TGF-β action, transcription factorUnknown
Rab3Membrane trafficVesicular traffic
Ubiquitin protein ligase E3Ubiquitin ligaseDegradation
SUMO-activating enzyme subunit 2E1-ligase for SUMO1/2/3Degradation
Cullin-associated NEDD8-dissociated protein 1E3 ubiquitin ligase complexesDegradation
Exportin-1Nuclear export of proteinsUnknown
Exportin-2Nuclear export of proteinsUnknown
Importin-7Prevents activation of Ran-GTPaseUnknown
Lamina B1Nuclear structure and dynamicsNuclear association
Lamina-associated polypeptide 2Assembly of the nuclear lamina/nuclear organizationNuclear association
MyosinATP-dependent motor proteinUnknown
Table
Table 4. Protein detected MS of immuno-purified α1D-adrenoceptors. * Data from [125]; ** our data.
Table 4. Protein detected MS of immuno-purified α1D-adrenoceptors. * Data from [125]; ** our data.
ProteinFunctionPossible Role
* SNTB Syntrophin, β 1 and 2Actin-binding proteinAdaptor protein
* UTRN UtrophinComponent of cytoskeletonAdaptor protein
* ERLIN 2 ER lipid raft associated 2Endoplasmic reticulum-associated degradationUnknown
* GOPCGolgi-associated PDZ and coiled-coil motif containingAdaptor protein
* ERLIN 1Endoplasmic reticulum-associated degradationUnknown
* DTNA, Distrobrevin αCytoplasmic proteins bind to syntrophinAdaptor protein
* SNTA1. Syntrophin α 1Actin-binding protein, protein associated with dystrophinAdaptor protein
* ACTAActin α 2 smooth muscle aortaCell motility
* AMFRAutocrine motility factor receptorMembrane traffic
* CKAP4Cytoskeleton-associated proteinCell motility
** OPA1Dynamin-like 120 kDa protein, mitochondrialUnknown
** DNM2Dynamin-2Scission of newly formed vesicles
** EEF2Elongation factor 2Unknown
** CSE1L, XPO1, XPO7Exportin 1, 2 and 7Unknown
** XRCC5X-ray repair cross-complementing protein 5Unknown
** MTHFD1C-1-tetrahydrofolate synthase, cytoplasmicUnknown
** UBA2SUMO-activating enzyme subunit 2Unknown
** NUP93Nuclear pore complex protein Nup93Unknown
** DDX21Nucleolar RNA helicase 2Unknown
** MCM7DNA replication licensing factor MCM7Unknown

Share and Cite

MDPI and ACS Style

Alfonzo-Méndez, M.A.; Alcántara-Hernández, R.; García-Sáinz, J.A. Novel Structural Approaches to Study GPCR Regulation. Int. J. Mol. Sci. 2017, 18, 27. https://doi.org/10.3390/ijms18010027

AMA Style

Alfonzo-Méndez MA, Alcántara-Hernández R, García-Sáinz JA. Novel Structural Approaches to Study GPCR Regulation. International Journal of Molecular Sciences. 2017; 18(1):27. https://doi.org/10.3390/ijms18010027

Chicago/Turabian Style

Alfonzo-Méndez, Marco A., Rocío Alcántara-Hernández, and J. Adolfo García-Sáinz. 2017. "Novel Structural Approaches to Study GPCR Regulation" International Journal of Molecular Sciences 18, no. 1: 27. https://doi.org/10.3390/ijms18010027

APA Style

Alfonzo-Méndez, M. A., Alcántara-Hernández, R., & García-Sáinz, J. A. (2017). Novel Structural Approaches to Study GPCR Regulation. International Journal of Molecular Sciences, 18(1), 27. https://doi.org/10.3390/ijms18010027

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop