Heteromerization of GPR55 and cannabinoid CB2 receptors modulates signalling.

BACKGROUND AND PURPOSE: Heteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. EXPERIMENTAL APPROACH: The direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. KEY RESULTS: GPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors. CONCLUSIONS AND IMPLICATIONS: Heteromers, unique signalling units, form in HEK293 cells expressing GPR55 and CB2 receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.

The GPCR-associated sorting protein 1 regulates ligand-induced down-regulation of GPR55.

BACKGROUND AND PURPOSE: Many GPCRs, including the CB(1) cannabinoid receptor, are down-regulated following prolonged agonist exposure by interacting with the GPCR-associated sorting protein-1 (GASP-1). The CB(1) receptor antagonist rimonabant has also recently been described to be an agonist at GPR55, a cannabinoid-related receptor. Here we investigated the post-endocytic properties of GPR55 after agonist exposure and tested whether GASP-1 is involved in this process. EXPERIMENTAL APPROACH: We evaluated the direct protein-protein interaction of GPR55 with GASP-1 using (i) GST-binding assays and (ii) co-immunoprecipitation assays in GPR55-HEK293 cells with endogenous GASP-1 expression. We further tested the internalization, recycling and degradation of GPR55 using confocal fluorescence microscopy and biotinylation assays in the presence and absence of GASP-1 (lentiviral small hairpin RNA knockdown of GASP-1) under prolonged agonist [rimonabant (RIM), lysophosphatidylinositol (LPI)] stimulation. KEY RESULTS: We showed that the prolonged activation of GPR55 with rimonabant or LPI down-regulates GPR55 via GASP-1. GASP-1 binds to GPR55 in vitro, and this interaction was required for targeting GPR55 for degradation. Disrupting the GPR55-GASP-1 interaction prevented post-endocytic receptor degradation, and thereby allowed receptor recycling. CONCLUSION AND IMPLICATIONS: These data implicate GASP-1 as an important regulator of ligand-mediated down-regulation of GPR55. By identifying GASP-1 as a key regulator of the trafficking and, by extension, functional expression of GPR55, we may be one step closer to gaining a better understanding of this receptor in response to cannabinoid drugs. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors.

A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we highlight the emerging impact of these receptor on virus-mediated diseases.

Virally encoded 7TM receptors.

A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion, cellular reprogramming, tissue targeting or for cell entry. Through their efficient evolutionary machinery and through in vivo selection performed directly on the human cellular and molecular targets, virus have been able to optimize the encoded receptors for distinct pharmacological profiles to help in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum of either CC chemokines, US28 from human cytomegalovirus, or CXC chemokines, ORF74 from human herpesvirus 8. Nevertheless, US28 has been specifically optimized for recognition of the membrane bound chemokine, fractalkine, conceivably involved in cell-cell transfer of virus; whereas ORF74 among the endogenous CXC chemokines has selected angiogenic chemokines as agonists and angiostatic/modulatory chemokines as inverse agonists. ORF74 possess substantial cell-transforming properties and signals with high constitutive activity through the phospholipase C and MAP kinase pathways. Interestingly, transgenic expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.

Kinetics of ternary complex formation with fusion proteins composed of the A(1)-adenosine receptor and G protein alpha-subunits.

High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.

Tight association of the human Mel(1a)-melatonin receptor and G(i): precoupling and constitutive activity.

If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel(1a)-melatonin receptor activates G(i)-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase Cbeta; the latter on condition that G(q) is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel(1a) receptor in both intact cells and isolated membranes. This suggests that the Mel(1a) receptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel(1a) receptor with G(i). Because the receptor density is in the physiological range, constitutive activity is not an artifact arising from overexpression of the receptor. In addition, the following findings indicate that the Mel(1a) receptor forms a very tight complex with G(i) which can be observed both in the presence and absence of an agonist. 1) In intact cells and in membranes, high-affinity agonist binding is resistant to the destabilizing effect of guanine nucleotides. 2) The ability to bind an agonist with high affinity is preserved even after exposure of the cells to pertussis toxin, because a fraction of G(i) is inaccessible to the toxin in cells expressing Mel(1a) receptors (but not the A(1)-adenosine receptor, another G(i)-coupled receptor). 3) An antiserum directed against the Mel(1a) receptor coprecipitates G(i) even in the absence of an agonist. We therefore conclude that the Mel(1a) receptor is tightly precoupled and that its constitutive activity may play a role in pacing the biological clock, an action known to involve the melatonin receptors in the suprachiasmatic nucleus.

G protein antagonists.

Heterotrimeric G proteins couple membrane-bound heptahelical receptors to their cellular effector systems (ion channels or enzymes generating a second messenger). In current pharmacotherapy, the input to G protein-regulated signalling is typically manipulated by targeting the receptor with appropriate agonists or antagonists and, to a lesser extent, by altering second messenger levels, most notably by inhibiting phosphodiesterases that hydrolyse cyclic nucleotides. When stimulated, G proteins undergo a cycle of activation and deactivation in which the alpha-subunits and the betagamma-dimers sequentially expose binding sites for their reaction partners (receptors, guanine nucleotides and effectors, as well as regulatory proteins). These domains can be blocked by inhibitors and this produces effects that cannot be achieved by receptor antagonists. Here, the structural and mechanistic information on G protein antagonists is summarized and an outline of the arguments supporting the hypothesis that G proteins per se are also potential drug targets is provided.

Differential uncoupling of A1 adenosine and D2 dopamine receptors by suramin and didemethylated suramin (NF037).

Suramin analogues uncouple two Gi/Go-coupled receptors, the D2 dopamine receptor in rat striatum and the A1 adenosine receptor in human cerebral cortex, with distinct structure-activity relations. This discrepancy may reflect true differences in the affinity of the analogues for specific receptor/G protein complexes or may be attributable to differences in species or in the tissue source used. We addressed this question by using human embryonic kidney 293 cells that stably express the human A1 and rat A1 receptor and the human D2 receptor. Suramin is 10-fold more potent than its didemethylated analogue NF037 in inhibiting the interaction between G proteins and the rat A1 or human A1 receptor; in contrast, both compounds are equipotent in uncoupling the D2 receptor. These differences are observed regardless of whether (1) inhibition of high affinity agonist binding to the receptors or (2) agonist-stimulated GTPgammaS binding is used as readout, (3) the receptors are allowed to interact with the G protein complement in human embryonic kidney 293 cell membranes, or (4) the receptors are forced to interact with a defined G protein alpha subunit (i.e., after reconstituting pertussis toxin-treated membranes with exogenous rGi alpha-1). The apparent affinity of suramin depends in a linear manner on receptor occupancy, which shows that suramin and the receptor compete for the G protein. Finally, the affinity of the receptors for rGi alpha-1 (human A1 > rat A1 > human D2) is inversely correlated with the potency of suramin in uncoupling ternary complexes formed by these receptors and thus determines the selectivity of the suramin analogues for specific receptor/G protein tandems.

Gsalpha-selective G protein antagonists.

Suramin acts as a G protein inhibitor because it inhibits the rate-limiting step in activation of the Galpha subunit, i.e., the exchange of GDP for GTP. Here, we have searched for analogues that are selective for Gsalpha. Two compounds have been identified: NF449 (4,4',4",4'"-[carbonyl-bis[imino-5,1,3-benzenetriyl bis-(carbonylimino)]]tetrakis-(benzene-1,3-disulfonate) and NF503 (4, 4'-[carbonylbis[imino-3,1-phenylene-(2, 5-benzimidazolylene)carbonylimino]]bis-benzenesulfonate). These compounds (i) suppress the association rate of guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[gammaS]) binding to Gsalpha-s but not to Gialpha-1, (ii) inhibit stimulation of adenylyl cyclase activity in S49 cyc- membranes (deficient in endogenous Gsalpha) by exogenously added Gsalpha-s, and (iii) block the coupling of beta-adrenergic receptors to Gs with half-maximum effects in the low micromolar range. In contrast to suramin, which is not selective, NF503 and NF449 disrupt the interaction of the A1-adenosine receptor with its cognate G proteins (Gi/Go) at concentrations that are >30-fold higher than those required for uncoupling of beta-adrenergic receptor/Gs tandems; similarly, the angiotensin II type-1 receptor (a prototypical Gq-coupled receptor) is barely affected by the compounds. Thus, NF503 and NF449 fulfill essential criteria for Gsalpha-selective antagonists. The observations demonstrate the feasibility of subtype-selective G protein inhibition.

G protein coupling of the rat A1-adenosine receptor--partial purification of a protein which stabilizes the receptor-G protein association.

A membrane protein identified in cortical brain membranes and termed 'coupling cofactor', modulates G protein-coupling of the A1-adenosine receptor by reducing the catalytic efficiency of the receptor. Coupling cofactor traps the A1-adenosine receptor in the high affinity complex and, thus, is responsible for the resistance of high affinity A1-agonist binding to modulation by guanine nucleotides. In the present work, this effect was used for assaying the activity of coupling cofactor by reconstituting guanine-nucleotide resistant agonist binding to rat A1-adenosine receptors in detergent extracted brain membranes or in membranes from 293 cells after stable transfection with receptor cDNA. Coupling cofactor was partially purified from porcine brain membranes. The specific activity was modestly enriched (approximately 5-fold) after three chromatographic steps (DEAE-Sephacel, AcA34, MonoQ pH 8). Rechromatography of coupling cofactor over MonoQ at pH 7 resulted in a loss in specific activity if membranes of 293 cells but not if brain membranes were used as acceptor membranes. In addition, the molecular mass estimated by gel filtration decreased from > 150 kDa in the initial stage of purification to 40-30 kDa after this fourth chromatographic step. These two observations suggest that coupling cofactor requires an additional component that is present in brain membranes and is lost in later stages of purification. The activity of partially purified preparations of coupling cofactor activity relied also on the abundance of G protein alpha-subunits in the membrane. The activity on reconstitution with brain membranes or pertussis toxin pretreated 293 membranes was supported by addition of Gi alpha (rank order of protency: alpha i1 > alpha i3 > alpha i2) but not of G(o alpha). The selectivity for G protein alpha-subunits suggests that coupling cofactor may provide for an additional level of specificity in organizing receptor-G protein coupling.