MUTATIONS TO FORSKOLIN RESISTANCE RESULT IN LOSS OF ADRENOCORTICOTROPIN RECEPTORS AND CONSEQUENT REDUCTIONS IN LEVELS OF G-PROTEIN ALPHA-SUBUNITS

A family of mutants isolated from the Y1 mouse adrenal cell line on th e basis of their resistance to the growth inhibitory effects of forsko lin have an underlying mutation that affects the activity of adenylyl cyclase. As part of the mutant phenotype, adenylyl cyclase is partiall y resistant to activation by forskolin, completely insensitive to ACTH , and fully responsive to NaF; the levels of G(s) alpha and G(i) alpha in plasma membrane fractions are decreased; and the activity of G bet a/gamma is impaired. In the present study, we examine the basis for th e complex phenotype associated with forskolin resistance to better und erstand the factors that contribute to the regulation of adenylyl cycl ase activity. We demonstrate that the resistance of these mutants to A CTH results from the failure to express ACTH receptor transcripts. Tra nsfection of these mutants with a gene encoding the mouse beta(2)-adre nergic receptor led to the recovery of transformants with normal recep tor-G protein coupling and with increased levels of G(s) alpha and G(i ) alpha that approached those in parental Y1 cells. These beta(2)-adre nergic receptor transformants, nonetheless, remained resistant to fors kolin and ACTH. Two spontaneous Y1 mutants, Y6 and OS3, previously cha racterized as ACTH-resistant clones that failed to accumulate ACTH rec eptor transcripts, were shown to be forskolin resistant and to contain less G(s) alpha in membrane fractions, indicating that forskolin resi stance, failure to express the ACTH receptor, and the consequent reduc tion in G(s) alpha are closely linked. Expression of the human ACTH re ceptor in Y6 and OS3 cells restored ACTH-responsive adenylyl cyclase a ctivity and increased the level of G(s) alpha, but did not otherwise r everse the forskolin-resistant phenotype. Together, these results demo nstrate that mutations to forskolin resistance have downstream consequ ences that result in the loss of ACTH receptor expression and the cons equent reduction in levels of membrane-associated G alpha subunits. Th e results further suggest that G protein-coupled receptors may have a stabilizing influence on G alpha subunits associated with the cell mem brane. According to current models, forskolin activates adenylyl cycla se by forming a ternary complex with adenylyl cyclase and G(s) alpha. Our results suggest that this model may be incomplete and that an addi tional component, acting directly or indirectly, is required for optim al activation of adenylyl cyclase by forskolin.