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. 1992 Oct 1;89(19):8933–8937. doi: 10.1073/pnas.89.19.8933

Defective guanyl nucleotide-binding protein beta gamma subunits in a forskolin-resistant mutant of the Y1 adrenocortical cell line.

J Mitchell 1, J K Northup 1, B P Schimmer 1
PMCID: PMC50038  PMID: 1409589

Abstract

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.

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Selected References

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