Evidence for stimulation of adenylyl cyclase by an activated G(s) heterotrimer in cell membranes: an experimental method for controlling the G(s) subunit composition of cell membranes.

Heterotrimeric (alphabetagamma) G(s) mediates agonist-induced stimulation of adenylyl cyclase (AC). Cholera toxin (CTx) will ADP-ribosylate the alpha-subunit of G(s) (G(s)alpha). G(s)alpha-deficient cyc(-) membranes were "stripped" of Gbeta. When the stripped cyc(-) were incubated with G(s)alpha and/or Gbetagamma, each was incorporated into the membranes independently of the other. Both G(s)alpha and Gbetagamma had to be present in the membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate G(s)alpha, indicating that the membrane bound G(s) heterotrimer is a substrate for CTx, but the G(s)alpha subunit by itself is not. When G(s)alpha was completely and irreversibly activated with GTPgammaS and incorporated into stripped cyc(-), it was a poor substrate for CTx and a weak stimulator of AC unless Gbetagamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of G(s) heterotrimer that was formed in the membranes from GTPgammaS-activated G(s)alpha and Gbetagamma. These data suggest that AC is stimulated by an activated G(s) heterotrimer in cell membranes.

Chloride effects on Gs subunit dissociation. Fluoroaluminate binding to Gs does not cause subunit dissociation in the absence of chloride ion.

The stimulatory guanine nucleotide binding protein (Gs) is heterotrimeric ( alpha beta gamma), and mediates activation of adenylyl cyclase by a ligand-receptor complex. The alpha subunit of Gs (Gsalpha) has a guanine nucleotide binding site, and activation occurs when tightly bound GDP is displaced by GTP. Together, GDP and fluoroaluminate (AlF4-) form a transition state analog of GTP that activates Gs. The work of other investigators suggests that AlF4- causes subunit dissociation when it activates Gs. We have observed that in solution AlF4- did not cause Gs subunits to dissociate unless NaCl was also present. The effect of NaCl was concentration dependent (10-200 mM). Omitting F-, Al3+, or Mg2+ prevented the NaCl-induced dissociation of Gs subunits. Na2SO4 could not substitute for NaCl in causing subunit dissociation, but KCl could, suggesting that the anion was responsible for the effect. Gs subunit reassociation occurred when the concentration of Cl- was reduced even though the concentrations of AlF4- and Mg2+ were maintained. The absence of Cl- did not prevent AlF4- binding to Gsalpha. We have concluded that AlF4-, a ligand which is capable of activating G proteins, can bind to Gs in solution without causing subunit dissociation.

Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit.

The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of two distinct insulin-binding components in solubilized human placental membranes by radioimmunoassay using synthetic peptide and anti-synthetic peptide antibody.

A peptide corresponding to the 957-980 sequence of human placental insulin receptor precursor (HIRP) was synthesized and antisera were produced against the synthetic peptide. Anti-synthetic HIRP(957-980) serum HIR-27 was proved to cross-react with HIRP-related proteins in solubilized human placental membranes. A radioimmunoassay developed with the antiserum and synthetic peptide HIRP(957-980) enabled us to separate, in combination with gel filtration, two insulin-binding components in solubilized human placental membranes which conceivably correspond to the alpha 2 beta 2 and alpha beta structures of the placental insulin receptor. The two components were shown to be distinct in insulin-binding behavior depending on conditions of pH and ionic strength in the binding assay.