The membrane localization of the G protein alpha s subunit is not dependent on its TENIR sequence or effector domain.
The GS protein alpha subunit (alpha S) sequences conserved in non-myristoylated alpha subunits (TENIR, residues 369-373) or critical for adenylyl cyclase interaction were investigated as possible sites required for membrane localization. Substitutions were created by site-directed mutagenesis in which the TENIR residues were deleted from alpha S or added to the soluble, non-myristoylated alpha i1. After transfection, COS cells were separated by centrifugation into particulate and soluble fractions. Immunoblots showed that these substitutions did not change the localization: alpha S +/- TENIR in the particulate fraction, non-myristoylated alpha i1 +/- TENIR in the soluble fraction. The constitutively active alpha i/alpha S chimera (CH4A), containing four regions of alpha S sufficient for adenylyl cyclase activation, was mutated to prevent myristoylation (GA-CH4A). Immunoblots of transfected COS cell fractions showed CH4A in the particulate and GA-CH4A in the soluble fraction. While these regions did not lead to membrane localization, the soluble GA-CH4A could activate adenylyl cyclase in the intact cell and after reconstitution with cyc- membranes.
Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
M Y
MY
Degtyarev
A M
AM
Spiegel
T L
TL
Jones
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008251:Macaca; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases