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.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: INSIGHTS FROM INTACT CELL PHOSPHORYLATION AND RECRUITMENT ASSAYS*

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α_2A-adrenergic receptors (α_2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α_2AAR with EC₅₀ values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu⁴, Val⁷, Leu⁸, Val¹¹, and Ser¹², directly interacts with receptors, whereas residues such as Asp¹⁰, Tyr¹³, Ala¹⁶, Met¹⁷, Gly⁴⁷⁵, Val⁴⁷⁷, and Ile⁴⁸⁵ are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.     

Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor

The adenosine A_2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A_2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A_2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A_2A receptor prior to ER exit. Complex formation between the A_2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A_2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A_2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A_2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.     

Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein-coupled receptor trafficking

The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, beta(2)-adaptin (the beta-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and beta(2)-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated beta(2)-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in beta(2)-adaptin binding and LIELD (residues 376-380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to beta(2)-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-DeltaLIELD) or beta(2)-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the beta(2)-adrenergic receptor, while arrestin/beta(2)-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and beta(2)-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated beta(2)-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.     

Development of Functionally Selective,Small Molecule Agonists at Kappa Opioid Receptors

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.     

A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes

Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β₁ adrenergic receptor (β₁AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t_½ of 3.9 h) in cardiac myocytes. However, the β₁AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β₁AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β₁AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.     

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β₂-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β₂-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.     

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 ⁴²⁰AKKAA⁴²⁴ mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gα_q association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.     

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

βArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that βarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of βarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of βarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of βarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that βarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.     

17β-Estradiol Elevates cGMP and,via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

Rapid non-genomic effects of 17β-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17β-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17β-estradiol, but not 17α-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Iα, stimulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17β-estradiol-BSA conjugate, suggesting that 17β-estradiol acts at the extracellular face of the plasma membrane. We also show that 17β-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative γ-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17β-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the γ-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.     

Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.     

Formation of a Ternary Complex among NHERF1, β-Arrestin,and Parathyroid Hormone Receptor

β-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for β-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in β-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na⁺/H⁺ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, β-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and β-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, β-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with β-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and β-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing β-arrestin2 into close proximity to the PTHR, thereby facilitating β-arrestin2 recruitment after receptor activation.     

Internalization of the Human Nicotinic Acid Receptor GPR109A Is Regulated by Gi,GRK2, and Arrestin3

Nicotinic acid (niacin) has been widely used as a favorable lipid-lowering drug for several decades, and the orphan G protein-coupled receptor GPR109A has been identified to be a receptor for niacin. Mechanistic investigations have shown that as a G_i-coupled receptor, GPR109A inhibits adenylate cyclase activity upon niacin activation, thereby inhibiting free fatty acid liberation. However, the underlying molecular mechanisms that regulate signaling and internalization of GPR109A remain largely unknown. To further characterize GPR109A internalization, we made a construct to express GPR109A fused with enhanced green fluorescent protein (EGFP) at its carboxyl-terminal end. In stable GPR109A-EGFP-expressing HEK-293 cells, GPR109A-EGFP was mainly localized at the plasma membrane and was rapidly internalized in a dose- and time-dependent manner upon agonist stimulation. GPR109A internalization was completely blocked by hypertonic sucrose, indicating that GPR109A internalizes via the clathrin-coated pit pathway. Further investigation demonstrated that internalized GPR109A was recycled to the cell surface after the removal of agonist, and recycling of the internalized receptors was not blocked by treatment with acidotropic agents, NH₄Cl and monensin. Pertussis toxin pretreatment not only inhibited forskolin-induced cAMP accumulation and intracellular Ca²⁺ mobilization; it also significantly attenuated agonist-promoted GPR109A internalization. Moreover, RNA interference experiments showed that knockdown of GRK2 (G protein-coupled receptor kinase 2) and arrestin3 expression significantly impaired receptor internalization. Taken together, these results indicate that the agonist-induced internalization of GPR109A receptors is regulated by GRK2 and arrestin3 in a pertussis toxin-sensitive manner and that internalized receptor recycling is independent of endosomal acidification.     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

Crucial Positively Charged Residues for Ligand Activation of the GPR35 Receptor

GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the β-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the β-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.     

Biased Agonism as a Mechanism for Differential Signaling by Chemokine Receptors

Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical “redundancy.” Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, β-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or β-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.     

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.