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.     

Defining the Molecular Basis for the First Potent and Selective Orthosteric Agonists of the FFA2 Free Fatty Acid Receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.     

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.     

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.     

Receptor-independent Ambient pH Signaling by Ubiquitin Attachment to Fungal Arrestin-like PalF

The seven-transmembrane receptor PalH and its coupled, positive-acting arrestin-like protein PalF are key components of a molecular sensor that in Aspergillus nidulans and other ascomycete fungi mediates activation of an intracellular signaling cascade by alkaline ambient pH. PalF is ubiquitinated in an alkaline pH- and PalH-dependent manner. We show here that PalF assists the plasma membrane localization of PalH and that PalF overexpression slightly hypersensitizes the pathway to alkaline pH but does not bypass the need for the ambient pH signal receptor in signaling. In contrast, covalent attachment of Ub to PalF activates the signaling pathway under acidic pH conditions in which the pathway is normally inactive, demonstrating a positive role for ubiquitination. We further show that PalF acts upstream of, or in concert with, the Bro1 domain-containing pH signaling protein PalC, which is normally recruited to cortical structures likely to represent active pH signaling foci under neutral/alkaline pH conditions. In agreement with its pathway-activating consequences, expression of PalF-Ub also promotes PalC cortical recruitment under acidic conditions. Notably, our data establish that expression of PalF-Ub, at approximately physiological levels, in a null palH background leads to a considerable degree of signaling even in the complete absence of the receptor. Thus PalF ubiquitination is a key, perhaps the sole, molecular trigger required for transmitting the alkaline pH signal to the downstream elements of the pathway.     

Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.     

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070–12082). Here, we identify the mediators of CB1 receptor internalization and {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced signaling mechanism for CB1 wherein β-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-β-arrestin 1 upon {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569. These results demonstrate for the first time the involvement of β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.     

The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.     

Differential Functions of ApoER2 and Very Low Density Lipoprotein Receptor in Reelin Signaling Depend on Differential Sorting of the Receptors

ApoER2 and very low density lipoprotein (VLDL) receptor transmit the Reelin signal into target cells of the central nervous system. To a certain extent, both receptors can compensate for each other, and only the loss of both receptors results in the reeler phenotype, which is characterized by a gross defect in the architecture of laminated brain structures. Nevertheless, both receptors also have specific distinct functions, as corroborated by analyses of the subtle phenotypes displayed in mice lacking either ApoER2 or VLDL receptor. The differences in their function(s), however, have not been defined at the cellular level. Here, using a panel of chimeric receptors, we demonstrate that endocytosis of Reelin and the fate of the individual receptors upon stimulation are linked to their specific sorting to raft versus non-raft domains of the plasma membrane. VLDL receptor residing in the non-raft domain endocytoses and destines Reelin for degradation via the clathrin-coated pit/clathrin-coated vesicle/endosome pathway without being degraded to a significant extent. Binding of Reelin to ApoER2, a resident of rafts, leads to the production of specific receptor fragments with specific functions of their own and to degradation of ApoER2 via lysosomes. These features contribute to a receptor-specific fine tuning of the Reelin signal, leading to a novel model that emphasizes negative feedback loops specifically mediated by ApoER2 and VLDL receptor, respectively.     

Distinct Roles of Arrestin 1 Protein in Photoreceptors during Drosophila Development

Arrestin regulates many facets of G-protein coupled receptor signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. Here we generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. We show that Arr1-eGFP is localized in the cytoplasm and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated rhodopsin 1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared with that of Arr2-eGFP. This slower kinetic activity may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, we show that in flies with reduced Arr1 prolonged depolarizing afterpotential can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation-deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors we failed to observe endocytosis of Arr1-eGFP. In contrast, we show that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, we propose that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae whereas it regulates the deactivation in adult.     

Comparative fluorescence resonance energy transfer analysis of metabotropic glutamate receptors: implications about the dimeric arrangement and rearrangement upon ligand bindings

Dimerization of G protein-coupled receptors has received much attention as a regulatory system of physiological function. Metabotropic glutamate receptors (mGluRs) are suitable models for studying the physiological significance of G protein-coupled receptor dimers because they form constitutive homodimers and function through dimeric rearrangement of their extracellular ligand binding domains. However, the molecular architecture of the transmembrane domains (TMDs) and their rearrangement upon agonist binding are still largely unknown. Here we show that the two helix Vs are arranged as the closest part in the dimeric TMDs and change their positions through synergistic control by the binding of two glutamates. The possibility that helix V is involved in an inter-protomer communication was first suggested by the finding that constitutively active mutation sites were identified on both sides of helix V. Then, comprehensive fluorescence resonance energy transfer (FRET) analysis using mGluRs whose cytoplasmic loops were labeled with donor and acceptor fluorescent proteins revealed that the third intracellular loop connecting helices V and VI of one protomer was in close proximity to the second and third intracellular loops of the other protomer and that all the intracellular loops became closer during the activation. Furthermore, FRET analysis of heterodimers in which only one protomer had ligand binding ability revealed the synergistic effect of the binding of two glutamates on the dimeric rearrangements of the TMD. Thus, the glutamate-dependent synergistic relocation of the helix Vs in the dimer is important for the signal flow from the extracellular ligand binding domain to the cytoplasmic surface of the mGluR.     

Silent scaffolds: inhibition OF c-Jun N-terminal kinase 3 activity in cell by dominant-negative arrestin-3 mutant

We established a new in vivo arrestin-3-JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3-luciferase and Venus-arrestins. We tested the ability of WT arrestin-3 and its 3A mutant that readily binds β2-adrenergic receptors as well as two mutants impaired in receptor binding, Δ7 and KNC, to directly bind JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding-deficient mutants interact with JNK3 significantly better than WT and 3A arrestin-3. WT arrestin-3 and Δ7 mutant robustly promoted JNK3 activation, whereas 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct arrestin functions. We found that the KNC mutant, which tightly binds ASK1, MKK4, and JNK3 without facilitating JNK3 phosphorylation, has a dominant-negative effect, competitively decreasing JNK activation by WT arrestin-3. Thus, KNC is a silent scaffold, a novel type of molecular tool for the suppression of MAPK signaling in living cells.     

High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs

Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, constitutive activity. To identify the molecular mechanisms responsible for this high constitutive activity, we have reconstituted a purified GHS-R1a monomer in a lipid disc. Using this reconstituted system, we show that the isolated ghrelin receptor per se activates G(q) in the absence of agonist, as assessed through guanosine 5'-O-(thiotriphosphate) binding experiments. The measured constitutive activity is similar in its extent to that observed in heterologous systems and in vivo. This is the first direct evidence for the high constitutive activity of the ghrelin receptor being an intrinsic property of the protein rather than the result of influence of its cellular environment. Moreover, we show that the isolated receptor in lipid discs recruits arrestin-2 in an agonist-dependent manner, whereas it interacts with μ-AP2 in the absence of ligand or in the presence of ghrelin. Of importance, these differences are linked to ligand-specific GHS-R1a conformations, as assessed by intrinsic fluorescence measurements. The distinct ligand requirements for the interaction of purified GHS-R1a with arrestin and AP2 provide a new rationale to the differences in basal and agonist-induced internalization observed in cells.     

Lgr4 Protein Deficiency Induces Ataxia-like Phenotype in Mice and Impairs Long Term Depression at Cerebellar Parallel Fiber-Purkinje Cell Synapses

Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4^−/− mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4^−/− mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4^−/− PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4^−/− cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia.     

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.