The melanosomal/lysosomal protein OA1 has properties of a G protein‐coupled receptor

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell‐specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein‐coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR‐mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well‐established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.     

Arrestins block G protein-coupled receptor-mediated apoptosis

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the beta(2)-adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.     

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.     

Arrestin-mediated signaling: Is there a controversy?

The activation of the mitogen-activated protein(MAP) kinases extracellular signal-regulated kinase(ERK)1/2 was traditionally used as a readout of signaling of G protein-coupled receptors(GPCRs) via arrestins, as opposed to conventional GPCR signaling via G proteins. Several recent studies using HEK293 cells where all G proteins were genetically ablated or inactivated, or both non-visual arrestins were knocked out, demonstrated that ERK1/2 phosphorylation requires G protein activity, but does not necessarily require the presence of non-visual arrestins. This appears to contradict the prevailing paradigm. Here we discuss these results along with the recent data on gene edited cells and arrestinmediated signaling. We suggest that there is no real controversy. G proteins might be involved in the activation of the upstream-most MAP3Ks, although in vivo most MAP3K activation is independent of heterotrimeric G proteins, being initiated by receptor tyrosine kinases and/or integrins. As far as MAP kinases are concerned, the best-established role of arrestins is scaffolding of the three-tiered cascades(MAP3K-MAP2 K-MAPK). Thus, it seems likely that arrestins, GPCRbound and free, facilitate the propagation of signals in these cascades, whereas signal initiation via MAP3K activation may be independent of arrestins. Different MAP3Ks are activated by various inputs, some of which are mediated by G proteins, particularly in cell culture, where we artificially prevent signaling by receptor tyrosine kinases and integrins, thereby favoring GPCR-induced signaling. Thus, there is no reason to change the paradigm: Arrestins and G proteins play distinct non-overlapping roles in cell signaling.     

The Effects of Ribavirin on the GTP Level and the VIP Receptor Dynamic of Human IGR39 Cells

The discovery that arrestins can function as ligand-regulated signaling scaffolds has revealed a previously unappreciated level of complexity in G protein-coupled receptor (GPCR) signal transduction. Because arrestin-bound GPCRs are uncoupled from G proteins, arrestin binding can be viewed as switching receptors between two temporally and spatially distinct signaling modes. Recent work has established two factors that underscore this duality of GPCR signaling and suggest it may ultimately have therapeutic significance. The first is that signaling by receptor-arrestin “signalsomes” does not require heterotrimeric G protein activation. The second is that arrestin-dependent signals can be initiated by pathway-specific “biased agonists,” creating the potential for drugs that selectively modulate different aspects of GPCR function. Currently, however, little is known about the physiological relevance of G protein-independent signals at the cellular or whole animal levels, and additional work is needed to determine whether arrestin pathway-selective drugs will find clinical application.     

Heptahelical terpsichory. Who calls the tune?

The discovery that arrestins can function as ligand-regulated signaling scaffolds has revealed a previously unappreciated level of complexity in G protein-coupled receptor (GPCR) signal transduction. Because arrestin-bound GPCRs are uncoupled from G proteins, arrestin binding can be viewed as switching receptors between two temporally and spatially distinct signaling modes. Recent work has established two factors that underscore this duality of GPCR signaling and suggest it may ultimately have therapeutic significance. The first is that signaling by receptor-arrestin "signalsomes" does not require heterotrimeric G protein activation. The second is that arrestin-dependent signals can be initiated by pathway-specific "biased agonists," creating the potential for drugs that selectively modulate different aspects of GPCR function. Currently, however, little is known about the physiological relevance of G protein-independent signals at the cellular or whole animal levels, and additional work is needed to determine whether arrestin pathway-selective drugs will find clinical application.     

Signaling through G protein coupled receptors

Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.Key words: heterotrimeric G proteins, GPCRs, seven-transmembrane receptors, signal transduction, stress signaling     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states

The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Implications of non-canonical G-protein signaling for the immune system

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which consist of three subunits α, β, and γ, function as molecular switches to control downstream effector molecules activated by G protein-coupled receptors (GPCRs). The GTP/GDP binding status of Gα transmits information about the ligand binding state of the GPCR to intended signal transduction pathways. In immune cells heterotrimeric G proteins impact signal transduction pathways that directly, or indirectly, regulate cell migration, activation, survival, proliferation, and differentiation. The cells of the innate and adaptive immune system abundantly express chemoattractant receptors and lesser amounts of many other types of GPCRs. But heterotrimeric G-proteins not only function in classical GPCR signaling, but also in non-canonical signaling. In these pathways the guanine exchange factor (GEF) exerted by a GPCR in the canonical pathway is replaced or supplemented by another protein such as Ric-8A. In addition, other proteins such as AGS3-6 can compete with Gβγ for binding to GDP bound Gα. This competition can promote Gβγ signaling by freeing Gβγ from rapidly rebinding GDP bound Gα. The proteins that participate in these non-canonical signaling pathways will be briefly described and their role, or potential one, in cells of the immune system will be highlighted.     

GPCR: G protein complexes—the fundamental signaling assembly

G protein coupled receptors (GPCR) constitute the largest group of cell surface receptors that transmit various signals across biological membranes through the binding and activation of heterotrimeric G proteins, which amplify the signal and activate downstream effectors leading to the biological responses. Thus, the first critical step in this signaling cascade is the interaction between receptor and its cognate G protein. Understanding this critical event at the molecular level is of high importance because abnormal function of GPCRs is associated with many diseases. Thus, these receptors are targets for drug development.     

Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.     

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.     

On the origins of arrestin and rhodopsin

Background  

G protein coupled receptors (GPCRs) are the most numerous proteins in mammalian genomes, and the most common targets of clinical drugs. However, their evolution remains enigmatic. GPCRs are intimately associated with trimeric G proteins, G protein receptor kinases, and arrestins. We conducted phylogenetic studies to reconstruct the history of arrestins. Those findings, in turn, led us to investigate the origin of the photosensory GPCR rhodopsin.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

G protein activation by serotonin type 4 receptor dimers: evidence that turning on two protomers is more efficient

The discovery that class C G protein-coupled receptors (GPCRs) function as obligatory dimeric entities has generated major interest in GPCR oligomerization. Oligomerization now appears to be a common feature among all GPCR classes. However, the functional significance of this process remains unclear because, in vitro, some monomeric GPCRs, such as rhodopsin and β(2)-adrenergic receptors, activate G proteins. By using wild type and mutant serotonin type 4 receptors (5-HT(4)Rs) (including a 5-HT(4)-RASSL) expressed in COS-7 cells as models of class A GPCRs, we show that activation of one protomer in a dimer was sufficient to stimulate G proteins. However, coupling efficiency was 2 times higher when both protomers were activated. Expression of combinations of 5-HT(4), in which both protomers were able to bind to agonists but only one could couple to G proteins, suggested that upon agonist occupancy, protomers did not independently couple to G proteins but rather that only one G protein was activated. Coupling of a single heterotrimeric G(s) protein to a receptor dimer was further confirmed in vitro, using the purified recombinant WT RASSL 5-HT(4)R obligatory heterodimer. These results, together with previous findings, demonstrate that, differently from class C GPCR dimers, class A GPCR dimers have pleiotropic activation mechanisms.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.