The tethered peptide activation mechanism of adhesion GPCRs.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell-cell and cell-extracellular matrix interactions1. Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers-N-terminal and C-terminal fragments-that remain non-covalently attached after receptors reach the cell surface1. Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood2-5. Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation.

GPR114/ADGRG5 is activated by its tethered peptide agonist because it is a cleaved adhesion GPCR.

Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAINA subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.

Hexahydroquinoline Derivatives Are Selective Agonists for the Adhesion G Protein-Coupled Receptor ADGRG1/GPR56.

GPR56 is a widely expressed adhesion GPCR (AGPCR) that has pleotropic roles in brain development, platelet function, cancer, and more. Nearly all AGPCRs possess extracellular regions that bind protein ligands and conceal a cryptic tethered peptide agonist. AGPCR reception of mechanical or shear force is thought to release the tethered agonist permitting its binding to the AGPCR orthosteric site for consequent activation of G protein signaling. This multistep mechanism of AGPCR activation is difficult to target, emphasizing the need for tool compounds and potential therapeutics that modulate AGPCRs directly. We expanded our cell-based pilot screen for GPR56 small molecule activators to screen >200,000 compounds and identified two promising agonists: 2-(furan-2-yl)-1-[(4-phenylphenyl)carbonyl]pyrrolidine, or compound 4, and propan-2-yl-4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, or compound 36. Both compounds activated GPR56 receptors enginered to have impaired tethered agonists and/or be cleavage deficient. Compound 4 activated a subset of group VIII AGPCRs while compound 36 had exclusive specificity for GPR56 among the GPCRs tested. Compound 36 SAR analysis identified an analog with the isopropyl R group replaced with a cyclopentyl ring and the electrophilic bromine replaced with a CF3 group. Analog 36.40 had 40% increased potency over compound 36 and was 20-fold more potent than synthetic peptidomimetics designed from the GPR56 tethered agonist. The new GPCR56 tool compounds discovered in this screen may be used to further advance understanding of GPR56 function and aid development of AGPCR-targeted therapeutics. SIGNIFICANCE STATEMENT: Adhesion G protein coupled receptors (AGPCRs) are a large, clinically relevant class of GPCRs with no available therapeutics, in part due to their unique mechanism of activation. GPR56 is a widely expressed model AGPCR involved in cancer metastasis, hemostasis, and neuron myelination. In the present study, we identified novel small molecule agonists for GPR56. These molecules are among the most potent identified thus far and may become useful leads in the development of a GPR56-targeted therapeutic.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56-/- mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Mechanisms of adhesion G protein-coupled receptor activation.

Adhesion G protein-coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein-coupling seven-transmembrane-spanning bundle. GAIN domain-mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

Production of Phosphorylated Ric-8A proteins using protein kinase CK2.

Resistance to Inhibitors of Cholinesterase-8 (Ric-8) proteins are molecular chaperones that fold heterotrimeric G protein α subunits shortly after biosynthesis. Ric-8 proteins also act as test tube guanine nucleotide exchange factors (GEF) that promote Gα subunit GDP for GTP exchange. The GEF and chaperoning activities of Ric-8A are regulated by phosphorylation of five serine and threonine residues within protein kinase CK2 consensus sites. The traditional way that Ric-8A proteins have been purified is from Spodoptera frugiperda (Sf9) or Trichoplusia ni (Tni) insect cells. Endogenous insect cell kinases do phosphorylate the critical regulatory sites of recombinant Ric-8A reasonably well, but there is batch-to-batch variability among recombinant Ric-8A preparations. Additionally, insect cell-production of some Ric-8 proteins with phosphosite alanine substitution mutations is proscribed as there seems to be interdependency of multi-site phosphorylation for functional protein production. Here, we present a method to produce wild type and phosphosite mutant Ric-8A proteins that are fully occupied with bound phosphate at each of the regulatory positions. Ric-8A proteins were expressed and purified from E. coli. Purified Ric-8A was phosphorylated in vitro with protein kinase CK2 and then re-isolated to remove kinase. The phosphorylated Ric-8A proteins were ∼99% pure and the completeness of phosphorylation was verified by chromatography, phos-tag SDS-PAGE mobility shifts, immunoblotting using phospho-site specific antibodies, and mass spectrometry analysis. E. coli-produced Ric-8A that was phosphorylated using this method promoted a faster rate of Gα subunit guanine nucleotide exchange than Ric-8A that was variably phosphorylated during production in insect cells.

Genetic Variant in Human PAR (Protease-Activated Receptor) 4 Enhances Thrombus Formation Resulting in Resistance to Antiplatelet Therapeutics.

OBJECTIVE: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. APPROACH AND RESULTS: Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. CONCLUSIONS: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.

Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5.

Adhesion G protein-coupled receptors (aGPCRs) have emerged as potential therapeutic targets in multiple cancers and in neurologic diseases. However, there are few modulatory compounds that act on these receptors. The majority of aGPCRs are orphans and a general activation mechanism has only recently been defined: aGPCRs are activated by a tethered agonist. aGPCRs constitutively cleave themselves during biosynthesis to generated two-part receptors comprising an extracellular domain (ECD) and a 7-transmembrane spanning domain (7TM). ECD dissociation reveals the tethered agonist initiating G protein signaling. Synthetic peptides that mimic the tethered agonist region can activate aGPCRs. We hypothesized that small molecules could act in the same way as peptide agonists. High throughput screening of the 2000-compound Spectrum Collection library using the serum response element luciferase gene reporter assay revealed two related classes of small molecules that could activate the aGPCR GPR56/ADGRG1. The most potent compound identified was 3-α-acetoxydihydrodeoxygedunin, or 3-α-DOG. 3-α-DOG activated engineered, low-activity GPR56 7TM in independent biochemical and cell-based assays with an EC50 of ∼5 µM. The compound also activated a subset of aGPCRs but not two class A GPCRs tested. The mode of 3-α-DOG-mediated receptor activation is that of partial agonist. 3-α-DOG activated GPR56 less efficaciously than peptide agonist and could antagonize both the peptide agonist and the endogenous tethered agonist, which are pharmacological hallmarks of partial agonists. Taken together, we have uncovered a novel group of aGPCR partial agonists that will serve as invaluable resources for understanding this unique class receptors.

Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.

The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.

Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector.

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCß. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or ß2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.

Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms.

Gαqßγ heterotrimer (Gq), an important mediator in the pathology of airway disease, plays a central role in bronchoconstriction and airway remodeling, including airway smooth muscle growth and inflammation. Current therapeutic strategies to treat airway disease include the use of muscarinic and leukotriene receptor antagonists; however, these pharmaceuticals demonstrate a limited clinical efficacy as multiple Gq-coupled receptor subtypes contribute to these pathologies. Thus, broadly inhibiting the activation of Gq may be an advantageous therapeutic approach. Here, we investigated the effects of broadly inhibiting Gq activation in vitro and ex vivo using receptor-dependent and receptor-independent strategies. P4pal-10 is a protease activated receptor 4-derived pepducin that exhibits efficacy toward multiple Gq-coupled receptors. Mechanistic studies demonstrated that P4pal-10 selectively inhibits all G protein coupling to several Gq-coupled receptors, including protease activated receptor 1, muscarinic acetylcholine M3, and histamine H1 receptors, while demonstrating no direct effect on Gq. We also evaluated the ability of FR900359, also known as UBO-QIC, to directly inhibit Gq activation. FR900359 inhibited spontaneous Gαq nucleotide exchange, while having little effect on Gαsßγ, Gαißγ, or Gα12/13ßγ heterotrimer activity. Both P4pal-10 and FR900359 inhibited Gq-mediated intracellular signaling and primary human airway smooth muscle growth, whereas only FR900359 effectively interdicted agonist-promoted airway contraction in human precision cut lung slices. These studies serve as a proof of concept that the broad-based inhibition of Gq activation may be a useful therapeutic approach to treat multiple common pathologies of airway disease.

Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits.

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gßγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.

The G protein α chaperone Ric-8 as a potential therapeutic target.

Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein-coupled receptors (GPCRs). G protein ßγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein-protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies.

M3 muscarinic receptor interaction with phospholipase C ß3 determines its signaling efficiency.

Phospholipase Cß (PLCß) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Gαßγ heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCß3. Both expressed and endogenous M3R interacted with PLCß in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCß3. Deletion of the PLCß3 C terminus or deletion of the PLCß3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCß3 plasma membrane localization. Purified PLCß3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCß3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Gαq-dependent but not Gßγ-dependent PLCß3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCß3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCß3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Gα.

Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.

Group II activators of G-protein signaling (AGS) serve as binding partners for Gα(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-Gα signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with Gα(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-Gα(i1)-YFP BRET were regulated by Ric-8A but not by Gα-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and Gα(i1)-YFP. The inhibitory regulation of GPR-Gα(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gα(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gα(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-G-Gα(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-Gα(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore Gα expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, Gα(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.

Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Ric-8 regulation of heterotrimeric G proteins.

Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.