Defective guanyl nucleotide-binding protein beta gamma subunits in a forskolin-resistant mutant of the Y1 adrenocortical cell line.
Forskolin-resistant mutants derived from Y1 adrenocortical cells display decreased responsiveness both to receptor and postreceptor stimulators of adenylyl cyclase and decreased amounts of the alpha subunits of the GTP-binding proteins (G proteins) that mediate stimulation (Gs) and inhibition (Gi) of adenylyl cyclase--namely, Gs alpha and Gi alpha-2. This phenotype is suggestive of a mutation that affects the processing or plasma membrane incorporation of G protein alpha subunits. Since the membrane attachment of heterotrimeric G proteins has been ascribed in part to the beta gamma subunits, we examined the quantity and functional activity of beta gamma subunits in wild-type Y1 and forskolin-resistant Forsk-10r-9 and Forsk-10r-3 cells. We now show that two assays previously used to examine the activity of purified beta gamma subunits--namely, to support either rhodopsin-catalyzed guanyl nucleotide exchange on Gt alpha or pertussis toxin-catalyzed ADP-ribosylation of Gt alpha--can be used with detergent extracts of cells. In both assays the beta gamma activity in Forsk-10r-9 and Forsk-10r-3 extracts was decreased by 53-76% compared with wild-type Y1 extracts. When normalized for immunoreactive beta subunit, the beta gamma activity in the Forsk-10r-9 samples was decreased by 55-57% compared with the wild-type Y1 samples. These results suggest that a mutation of one of the G protein beta or gamma subunits may result in the multiple defects of adenylyl cyclase activity and apparent loss of G protein alpha subunits seen in the forskolin-resistant mutant cells. The frequency with which these spontaneous mutations arise in the Y1 cell line suggests that they may contribute more generally to genetic abnormalities in signal transduction.
Mitchell
J
J
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
Northup
J K
JK
Schimmer
B P
BP
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000306:Adrenal Cortex Neoplasms; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D016296:Mutagenesis; D037342:Pertussis Toxin; D051381:Rats; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015293:Transducin