β₁ and β₂-adrenergic receptor expression in differentiating 3T3-L1 cells

Regulation of two highly homologous GTP-binding regulatory protein- (G-protein) linked receptors, β₁-and β₂-adrenergic receptors, was probed at the level of mRNA in differentiating 3T3-L1 cells. Expression of the two receptor subtypes at the protein level was defined by competition of radioligand binding with CGP-20712A, a highly selective β₁-adrenergic antagonist. 3T3-L1 fibroblasts express equivalent levels of β₁- and β₂-adrenergic receptors. Following treatment with dexamethasone and isobutylmethyl xanthine (IBMX), 3T3-L1 cells differentiate to adipocytes and express 4-fold more receptor, predominantly β₂-subtype (β₁-/β₂- ratio, 5:95). Regulation of β₁- and β₂-receptor mRNA levels by differentiation, as well as by steroid alone and IBMX alone was probed by DNA excess solution hybridization. A β₁-receptor antisense probe was constructed from double-stranded DNA assembled from synthetic oligonucleotides. In untreated 3T3-L1 fibroblasts the steady-state levels of β₁- and β₂-adrenergic receptor mRNA were equivalent (∼1.2 amol mRNA/μg total cellular RNA). β₂-Adrenergic receptor mRNA levels increased 3-fold as 3T3-L1 fibroblasts were differentiated to adiopcytes (day 7). mRNA levels for β₁-adrenergic receptor, in contrast, increased at day 2, but thereafter declined, falling to less than 0.05 amol mRNA/μg total cellular RNA by day 7 in adipocytes. A 7-day challenge with dexamethasone reduced by 50% β₁-adrenergic receptor mRNA levels. Treatment with IBMX alone reduced mRNA levels for both receptor subtypes. Neither steroid nor IBMX alone promoted differentiation. The present work, for the first time, demonstrates (i) the mRNA levels on a molar basis for two highly homologous G-protein-linked receptors expressed in a single cell, (ii) independent regulation of their mRNA levels that correlates well with receptor expression, and (iii), that it is differentiation in 3T3-L1 cells per se and not treatment with glucocorticoid or IBMX alone that promotes the up-regulation of the β₂-receptor transcripts and down-regulation of β₁-receptor transcripts.