Molecular complexes involved in the regulation of adenylate cyclase.

Abstract

Selective extraction of the adenylate cyclase regulatory protein (N-protein) from pigeon erythrocyte plasma membranes provided evidence for its cytoskeletal association. Cholate, but not Triton X-100 or digitonin, was effective in solubilizing the ADP-ribosylated N-protein. The labeled protein complex or components thereof that were associated with the Triton-insoluble cytoskeleton (shells) could be partly released by 0.1 mM EDTA; 1 M KCI in the presence of Triton X-100 achieved complete solubilization. 5'-Guanylyl imidodiphosphate (p[NH]ppG) and NaF, activators of adenylate cyclase, promoted the release of the regulatory protein from the cytoskeleton but MnC12, an "uncoupler" of the adenylate cyclase system, had the opposite effect. The solubilized, labeled N-protein was able to bind specificially to rat eryth-ocyte inside-out vesicles in the presence of divalent cations. A proteolytic product of inside-out vesicles inhibited the binding of the N-protein to fresh vesicles. Three molecular species which contained the Mr 45,000 polypeptide component of the N-protein were identified by gel permeation chromatography and by sucrose density gradient velocity sedimentation. p[NH]ppG appeared to convert the two larger molecular complexes to a smaller molecular entity. Such a molecular dissociation might be relevant to the effects of guanyl nucleotides on the activity of adenylate cyclase and on the affinity of hormone receptors.Activation ofthe adenylate cyclase system by hormones or GTP analogs appears to involve changes in the molecular associations among the receptor, the regulatory nucleotide-binding protein (N-protein), and the catalytic component. Gel permeation chromatography (1), sucrose density gradient sedimentation (2, 3), and target-irradiation analysis (4) have provided evidence for these molecular changes. Furthermore, selective extraction (5) and reconstitution (6) experiments suggested that constituents of the adenylate cyclase complex were bound to the cytoskeleton in a manner that depended on the state of activation of the enzyme (6). This report focuses on several molecular interactions of the N-protein and on their regulation by agents that activate adenylate cyclase. We have also attempted to solubilize, separate, and characterize the putative molecular complexes that result from these interactions. This approach may help to elucidate further the mechanism of activation of the enzyme as well as the mechanism of the regulation ofthe number and the affinity of hormone receptors that act on adenylate cyclase.Our (125-150 g) and from White Carneau pigeons. Rat reticulocytes were produced by the phenylhydrazine injection method (1). Pigeon erythrocytes were lysed by freeze-thawing, and the plasma membrane fraction was separated as reported (9). Rat reticulocyte and erythrocyte ghosts were made by hypotonic lysis (10) and they were resealed in the presence of MgCl2 (11). Inside-out vesicles were prepared by incubation of the erythrocyte ghosts in 0.1 mM sodium phosphate buffer (pH 7.6) at 37°C ...

Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.

“…We had reported previously the existence of a functionally relevant interaction between membrane-associated components of the adenylate cyclase system and erythrocyte cytoskeletons prepared by detergent extraction (18,19,22). Here we show that the stimulatory effect of the cytosolic factor persisted when detergent extraction followed the activation step.…”

Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: Digitonin-insoluble β-adrenergic receptor, adenylate cyclase, and cholera toxin substrate

“…The 45,000 tool wt (45K) polypeptide cholera toxin substrate, a subunit of the putative guanylnucleotide regulatory component, has been shown by Triton X-100 extraction and direct binding studies to be functionally associated with the cytoskeleton in rat and human erythrocytes [28,30]. The weaker detergent, digitonin, fails to remove an appreciable amount (less than 15%) of the 45K polypeptide from either rat reticulocytes or erythrocytes or pigeon erythrocyte membranes under the standard conditions described in this communication, which include the presence of serine protease inhibitors at 0 ~ (Fig.…”

“…Interactions among these components have been determined by a series of genetic complementations and the use of several reconstitution systems [13,17,27,33]. Recent work in this laboratory has involved the construction of a reconstitution system to investigate the interaction of the three components of the hormone-sensitive adenylate cyclase system with other protein and lipid components of the rat erythrocyte plasma membrane [20,[28][29][30]. Retention of the N-protein and the catalytic adenylate cyclase in cytoskeletal matrices generated with the nonionic detergent, Triton X-100, along with extraction and rebinding experiments under activating conditions, characterized distinct proteinaceous binding sites for these proteins.…”

Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin.

“…There are three (40, 53; 54) and perhaps five (55) a subunits and pertussis toxin substrates of similar molecular mass (39,40, and 41 kDa) in the brain; two (39 and 41 kDa) have been designated G. and G1, respectively (39,45,47,56), and there appear to be two forms of G. (57).…”

“…A possible reason for the small amount of G, in the PSD fraction is that the 0.5% Triton X-100 detergent treatment of SM, the first step in the liberation of PSD from the SM (1), solubilized some of the G protein from the PSD, since it was found (45) that Triton X-100 released substrates for cholera toxinactivated ADP-ribosylation from erythrocyte membrane (47) and released substrates for pertussis toxin-activated ADPribosylation from heart membrane (48). Also, the small amount of G1 in the PSD may be due to contamination by membranes.…”