Agonist regulation of β-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants

Agonist-promoted down-regulation of β-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of β-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 ± 0.08 (n = 8) amol/μg total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the β-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT₁ MF-2 cells (Hadcock, J.R., and Malbon, C.C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in G(sα) (H21a, unc, cyc^-) and a single variant lacking cAMP-dependent protein kinase activity (kin^-). H21a (G(sα) coupled to receptor, but not to adenylate cyclase), unc (G(sα) uncoupled from receptor), and cyc^- (lacking G(sα)) variants accumulated cAMP and down-regulated βAR mRNA in response to forskolin. In unc and cyc^- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of β-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin^- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin^-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-G(s) coupling is required (H21a), and clones lacking G(sα) (cyc^-) or receptor G(s) coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-G(s) coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.