Pharmacological analysis of a dopamine D(2Short):G(alphao) fusion protein expressed in Sf9 cells

A dopamine D(2Short) receptor:G(alphao) fusion protein was expressed in Sf9 cells using the baculovirus expression system. [(3)H]Spiperone bound to D(2Short):G(alphao) with a pK(d) approximately 10. Dopamine stimulated the binding of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) to D(2Short):G(alphao) expressed with Gbeta(1)gamma(2) (E(max)>460%; pEC(50) 5.43+/-0.06). Most of the putative D(2) antagonists behaved as inverse agonists (suppressing basal [(35)S]GTPgammaS binding) at D(2Short):G(alphao)/Gbeta(1)gamma(2) although (-)-sulpiride and ziprasidone were neutral antagonists. Competition of [(3)H]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapomorphine revealed two binding sites of different affinities, even in the presence of GTP (100 micro M). The D(2Short):G(alphao) fusion protein is therefore a good model for characterising D(2) receptors.     

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin-sensitive and insensitive G-proteins

We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G-proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist-mediated stimulation of [(35)S]GTP gamma S binding. In the present study, we observed that G(i/o)-type G-protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT-induced [(35)S]GTP gamma S binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT-mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U-73122, we showed that both signaling pathways contribute to the NT-mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx-sensitive and insensitive G-proteins.     

Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.

Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.     

Regulation of the coupling to different G proteins of rat corticotropin-releasing factor receptor type 1 in human embryonic kidney 293 cells

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC(50) values as measured by stimulation of [(35)S]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [(35)S]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [(35)S]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11)in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i) -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.     

Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11alpha following fusion to the alpha1b-adrenoreceptor: palmitoylation of G11alpha is not required for interaction with beta*gamma complex.

Transfection of either the alpha(1b)-adrenoreceptor or Galpha(11) into a fibroblast cell line derived from a Galpha(q)/Galpha(11) double knockout mouse failed to produce elevation of intracellular [Ca(2+)] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the alpha(1b)-adrenoreceptor with the palmitoylation-resistant C9S,C10S Galpha(11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Galpha(11) or C10S Galpha(11). Expression of a fusion protein between the alpha(1b)-adrenoreceptor and Galpha(11) allowed [Ca(2+)](i) elevation, and this was also true for a fusion protein between the alpha(1b)-adrenoreceptor and C9S,C10S Galpha(11), since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin alpha, as a beta.gamma-sequestering agent, fully attenuated the Ca(2+) signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Galpha(11) were also targets for agonist-regulated [(3)H]palmitoylation and bound [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [(35)S]GTPgammaS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release beta.gamma complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca(2+) signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta.gamma complex.     

The receptor-Galpha fusion protein as a tool for ligand screening: a model study using a nociceptin receptor-Galphai2 fusion protein

As a model system to screen endogenous ligands for G(i)-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and alpha subunit of G(i2). We detected nociceptin binding to the fusion protein by measuring stimulation of [(35)S]GTPgammaS binding with an EC(50) of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [(35)S]GTPgammaS binding to the fusion protein was clearly observed in the presence of an appropriate concentration of GDP, because the affinity for GDP was decreased in the presence of agonist. Full and partial agonists differed in their effects on apparent the affinity of the fusion protein for GDP: the IC(50) values for GDP to displace 100 pM [(35)S]GTPgammaS were estimated to be 2 micro M, 0.4 micro M, and 0.05 micro M in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [(35)S]GTPgammaS binding to the fusion protein in the brain extract derived from 2-3 g wet weight tissue without false-positive results. The active component was identified as endogenous nociceptin itself. These results indicate that the fusion protein of GPCR and Galpha(i) is useful for screening of endogenous ligands.     

Phosphorylation by protein kinase C modulates agonist binding to striatal dopamine D2 receptors

The effect of purified protein kinase C (PKC) on dopamine D2 receptor binding was studied. Saturation binding with [3H]spiperone was not affected. In competition experiments using agonists PKC-treated membranes showed a significant reduction in the proportion of high affinity sites, and the influence of GTP gamma S was abolished. These results suggest that PKC-dependent mechanisms can regulate the coupling between the dopamine D2 receptor and its G-protein.     

Solubilization and characterization of leukotriene B4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B4 (LTB4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with Kd and Bmax values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB4, and Ki values for 20-hydroxy- and 20-carboxy-LTB4, both inactive metabolites of LTB4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 microM GTP gamma S, a low affinity binding site appeared with a Kd value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTP gamma S on LTB4 binding, indicating that the LTB4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.     

Hypocretin stimulates [(35)S]GTP gamma S binding in Hcrtr 2-transfected cell lines and in brain homogenate

In vitro functional analyses of hypocretin/orexin receptor systems were performed using [(125)I]hypocretin radioreceptor and hypocretin-stimulated [(35)S]GTP gamma S binding assay in cell lines expressing human or canine (wild-type and narcoleptic-mutation) hypocretin receptor 2 (Hcrtr 2). Hypocretin-2 stimulated [(35)S]GTP gamma S binding in human and canine Hcrtr 2 expressing cell lines, while cell lines expressing the mutated canine Hcrtr 2 did not exhibit specific binding for [(125)I]hypocretin or hypocretin-stimulated [(35)S]GTP gamma S. In rat brain homogenates, regional specific hypocretin-stimulated [(35)S]GTP gamma S binding was also observed. Hypocretin-stimulated [(35)S]GTP gamma S binding, may thus be a useful functional assay for hypocretin receptors in both cell lines and brain tissue homogenates.     

Engineering a V2 Vasopressin Receptor Agonist- and Regulator of G-Protein-Signaling-Sensitive G Protein

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.     

Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).     

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.