A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 1986
The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different alpha chains. The family includes Gs and Gi, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; Go, a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine alpha t1 cDNA as a hybridization probe, we have isolated mouse cDNAs that encode alpha chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as alpha s because it specifically fails to hybridize with any transcript in an alpha s-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be alpha i. These alpha chains and those of the retinal transducins exhibit impressive sequence homology. Of the four, alpha t1 and alpha t2 are most alike (81% identical amino acid residues), whereas the presumptive alpha i is more similar than alpha s to alpha t1 (63% vs. 38% identical residues). Sequence homologies with p21ras and elongation factor Tu identify regions of the alpha chains that form the site for GTP binding and hydrolysis. Further comparison of the alpha-chain sequences suggests additional regions that may contribute to interactions with beta gamma subunits and the receptor and effector components of different signal transduction systems.
Biochemistry, 1990
An antibody (RM) raised against the carboxyl-terminal decapeptide of the a subunit of the stimulatory guanine nucleotide regulatory protein (Gsa) has been used to study the interaction of Gsa with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11. R M antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-7-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-y-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsa protein in the partially purified preparations was determined by immunoblotting with R M antibody. There was 3-fold more G s a detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with R M antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gscu protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goa, Gia, and GO. The GO protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of GP was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the R M antibody immunoprecipitates from control and GTP-7-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsa protein can be isolated by immunoprecipitation with an anti-Gsa antibody. There does not appear to be a specific association of Goa, Gia, or GP with the preactivated complex of the catalytic subunit and the activated Gsa subunit. Hormone-sensit ive adenylate cyclase is regulated by the interaction of hormone receptors with specific guanine nucleotide binding proteins (G proteins), which transduce the hormone signal to the adenylate cyclase catalytic subunit (Gilman, 1987; Spiegel, 1987). G proteins are heterotrimeric proteins consisting of a, 0, and y subunits. The a subunits
A surface on the G protein -subunit involved in interactions with adenylyl cyclases
Proceedings of the National Academy of Sciences, 1997
Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G to identify residues on G that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G86-105) and 115-135 (G115-135) from G. The G86-105 peptide inhibited G␥ stimulation of AC2 and blocked G␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G115-135 peptide were similar to those of G86-105 but G115-135 was less efficacious in blocking G␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G as a surface that is involved in transmitting signals to effectors.
The regulation of adenylyl cyclase by receptor-operated G proteins
Pharmacology & Therapeutics, 1991
The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (R s and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors
Proceedings of the National Academy of Sciences, 1999
The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with ␥ subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe ␥-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G s␣ (G s␣ *) or Ca 2؉ ͞ calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by ␥ subunits, the presence of both of these regions formed another ␥ interaction site that was sufficient to observe G s␣ *-or Ca 2؉ ͞CaM-stimulated activity. Inhibition of AC activity attributable to interaction of ␥ subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of ␥ on various effectors. Moreover, the C1 region of ACI was sufficient to observe G i␣1 -elicited inhibition of Ca 2؉ ͞CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G i␣1 , the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory inf luences of G i␣1 are mediated on the C1 regions of both ACI and ACV. The effects of ␥ on ACI can be mediated by interactions with the C1a region and a ␥ interacting site formed by the C1b and C2 domains of this enzyme.
Different roles for Gi and Go proteins in modulation of adenylyl cyclase type-2 activity
Journal of Neurochemistry, 2002
The effect of G i/o protein-coupled receptors on adenylyl cyclase type 2 (AC2) has been studied in Sf9 insect cells. Stimulation of cells expressing AC2 with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to a twofold stimulation of cAMP synthesis that could be blocked with the protein kinase C inhibitor GF109203X. Activation of a coexpressed a 2A -adrenoceptor or muscarinic M4 receptor inhibited the stimulation by TPA almost completely in a pertussis toxinsensitive manner. Activation of G s proteins switched the response of the a 2A -adrenoceptor to potentiation of prestimulated AC2 activity. The potentiation, but not the inhibition, could be blocked by a Gbc scavenger. A novel methodological approach, whereby signalling through endogenous G proteins was ablated, was used to assess specific G protein species in the signal pathway. Expression of G o proteins (a o1 + b 1 c 2 ) restored both the inhibition and the potentiation, whereas expression of G i proteins (a i1 + b 1 c 2 ) resulted in a potentiation of both the TPA-and the G s -stimulated AC2 activity. The data presented supports the view of AC2 as a molecular switch and implicates this isoform as a target for G o protein-linked signalling.
A surface on the G protein β-subunit involved in interactions with adenylyl cyclases
Proceedings of the National Academy of Sciences, 1997
Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G to identify residues on G that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G86-105) and 115-135 (G115-135) from G. The G86-105 peptide inhibited G␥ stimulation of AC2 and blocked G␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G115-135 peptide were similar to those of G86-105 but G115-135 was less efficacious in blocking G␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G as a surface that is involved in transmitting signals to effectors.
Adenylyl Cyclase Regulates Signal Onset via the Inhibitory GTP-binding Protein, Gi
Journal of Biological Chemistry, 2000
Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G s and G i , respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for G␣ s and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G s (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328 -1331). This latter action of ACV would facilitate the rapid onset of signaling via G s . Because the C1 region of ACV interacts with the inhibitory GTP-binding protein G␣ i , we investigated whether the receptor-mediated activation of heterotrimeric G i was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G i such that the amount of peptide required to stimulate G i in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5-(␥-thio)triphosphate (GTP␥S) to G i was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTP␥S binding to G i in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc ؊ cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G i and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G i .
Regions in the G Protein Subunit Important for Interaction with Receptors and Effectors
Molecular Pharmacology, 2005
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.