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.
Ganpat
M M
MM
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4440, USA.
Nishimura
M
M
Toyoshige
M
M
Okuya
S
S
Pointer
R H
RH
Rebois
R V
RV
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D003584:Cytological Techniques; D003902:Detergents / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D000276:Adjuvants, Immunologic; D003902:Detergents; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008565:Membrane Proteins; D000077423:Polidocanol; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011092:Polyethylene Glycols; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases