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Decreased Levels of Guanyl Nucleotide-Binding Regulatory Protein -Subunits in Y1 Adrenocortical Tumor Cell Mutants Resistant to Forskolin

Banting and Best Department of Medical Research, University of Toronto Toronto, Ontario, Canada M5G 1L6

Address requests for reprints to: Dr. Bernard P. Schimmer, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.

Abstract

Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'- yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the -subunits of Gs and Gi-2 were reduced by 70–80% relative to the levels in parental Y1 cells. The levels of the β₃₆-subunit were much less affected, and the levels of the i-3 and β₃₅-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs and Gi RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs and Gi-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G subunits or their incorporation into the plasma membrane.

FOOTNOTES

This work was supported by a research grant from the Medical Research Council of Canada.

Received for publication June 26, 1990. Revision received August 13, 1990. Accepted for publication August 20, 1990.

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