Antibodies as probes of G-protein receptor-effector coupling and of G-protein membrane attachment.
The specificity of antisera raised against synthetic decapeptides corresponding to the C-terminus of G-protein alpha-subunits was rigorously defined. Antisera raised against alpha-subunit C-terminal decapeptides proved capable of immuno-precipitating their cognate G-proteins, as well as recognizing these proteins in native cell membranes. Thus the alpha s-specific antiserum could block agonist-stimulated adenylyl cyclase activity in native membranes and also immuno-precipitate an activated alpha s-adenylyl cyclase complex. The alpha i 2-, but not alpha i 3- and alpha z-specific antiserum could block agonist-mediated inhibition of adenylyl cyclase in human platelet membranes. These results indicate that the C-terminal decapeptide is involved in G-protein receptor, but not effector, coupling. These antisera also proved useful in immunoprecipitation of endogenous and transfected alpha-subunits in COS cells. Using this approach, we were able to show that both alpha s and alpha i are membrane associated, but only the latter is myristylated. A mutant alpha i 1 (second residue Gly changed to Ala) fails to incorporate myristate and is localized in the soluble fraction. Myristylation is thus essential for membrane attachment of alpha i, but not alpha s.
Spiegel
A M
AM
Molecular Pathophysiology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Simonds
W F
WF
Jones
T L
TL
Goldsmith
P K
PK
Unson
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000906:Antibodies; D008024:Ligands; D015335:Molecular Probes; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins