The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively.

Many a subuts of heterotrimeric guanine nudeotide-binding regulatory proteins (G proteins) A family ofguanine nucleotide-binding regulatory proteins (G proteins) serves in signal transduction systems to link receptors exposed at the cell surface to intracellular effectors. Upon activation by receptor, a heterotrimeric G protein dissociates into a GTP-bound a subunit and a 83'y subunit complex, which are then able to modulate effector activity. The structurally homologous a subunits of the Gi subfamily (a;, a., and a) are modified by myristate (C14:0), bound in amide linkage at their amino-terminal glycine residues (1, 2). Retinal transducin a subunit (at) is heterogeneously modified at the same site by myristate and by C14:1, C14:2, and C12:0 fatty acids (3, 4). Myristoylation increases the apparent affinity of modified a subunits for 13y and for effector and appears to facilitate association of a with cellular membranes (2, 5-7). Some G protein a subunits, including G. and Gq, are not myristoylated. However, members of the G, and Gq subfamilies of a subunits are palmitoylated (C16:0) (8). In addition, members of the G, subfamily (with the exception of at) are both palmitoylated and myristoylated at separate sites (8). We have proposed that Cys-3 is the site of palmitoylation of members of the G. and Gi subfamilies.Palmitoylation can be a dynamic posttranslational modification of proteins, unlike myristoylation, which is usually an irreversible cotranslational modification (9). We have speculated that a cycle ofacylation and deacylation could regulate the activity and membrane association of G-protein a subunits (8). As initial steps to test this hypothesis, we have examined receptor-dependent changes in the palmitoylation of as, and we have tested the capacity of mutant a subunits that cannot be palmitoylated to associate with membranes.

Various heterotrimeric guanine nucleotide-binding proteins have been identified on the basis of the individual subtypes of their alpha subunits. The beta gamma complexes, composed of beta and gamma subunits, remain tightly associated under physiological conditions and have been assumed to constitute a common pool shared among various guanosine triphosphate (GTP)-binding (G) protein heterotrimers. Particular alpha and beta subunit subtypes participate in the signal transduction processes between somatostatin or muscarinic receptors and the voltage-sensitive L-type calcium channel in rat pituitary GH3 cells. Among gamma subunits the gamma 3 subtype was found to be required for coupling of the somatostatin receptor to voltage-sensitive calcium channels, whereas the gamma 4 subtype was found to be required for coupling of the muscarinic receptor to those channels.

Forskolin-and G s␣ -stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ϳ25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M r ϳ 120,000). The C 1a domain from type V adenylyl cyclase (VC 1 ) and the C 2 domain from type II adenylyl cyclase (IIC 2 ) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 mol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC 1 , IIC 2 , and guanosine 5-O-(3-thiotriphosphate) (GTP␥S)-G s␣ is responsible for the observed enzymatic activity and can be isolated. In addition, GTP␥S-G s␣ interacts with homodimers of IIC 2 to form a heterodimeric complex (one molecule each of G s␣ and IIC 2 ) but not detectably with homodimers of VC 1 . Nevertheless, G s␣ can be cross-linked to VC 1 in the activated heterotrimeric complex of VC 1 , IIC 2 , and G s␣ , indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C 1 and C 2 .Eleven distinct isoforms of mammalian adenylyl cyclase have been identified to date, and the regulatory properties of several of these proteins have been characterized extensively (1, 2). Although there is remarkable variation in the responsiveness of these enzymes to inhibitory effects of the G i␣ 1 proteins and to such agents as G protein ␤␥ subunits and Ca 2ϩ , the catalytic activity of all of the known isoforms is stimulated by the ␣ subunit of G s and, presumably nonphysiologically, by the diterpene forskolin. The adenylyl cyclases share a unique structure for an enzyme, resembling transporters such as the P-glycoproteins topographically. They are intrinsic membrane proteins by virtue of their two large hydrophobic domains, each of which is hypothesized to contain six membrane-spanning helices. The first of these hydrophobic regions follows a short amino-terminal sequence and precedes a roughly 40-kDa cytoplasmic domain (C 1 ). The second hydrophobic region separates C 1 from a second cytosolic domain (C 2 ) of comparable size. Each of the two cytosolic domains includes a sequence of 200 -250 amino acid residues that is typically 50% similar to its consort, 50 -90% similar to the corresponding domains of other adenylyl cyclase isoforms, and 20 -25% similar to the catalytic domains of membrane-bound and cytosolic guanylyl cyclases.Detailed biochemical characterization of adenylyl cyclase is impaired by the insolubility, instability, and sparsity of the native enzyme, as well as our incapacity to express necessary amounts of the protein in heterologous systems. To overcome these hurdles we have synthesized (...

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.