Down-regulation of beta-adrenergic receptors: agonist-induced reduction in receptor mRNA levels.

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Abstract
Incubation of DDT1 MF-2 hamster vas deferens cells with .beta.-adrenergic agonists results in a time- and concentration-dependent decrease in both .beta.-adrenergic receptor (.beta.AR) responsiveness and receptor number. Receptor mRNA levels were quantified by DNA-excess solution hybridization by using a 170-nucleotide single-stranded probe derived from the hamster .beta.2AR cDNA. RNA blot analysis of poly(A)+-selected RNA with the solution probe revealed a 2.2-kilobase species. Digestion of the RNA/solution probe mixture with S1 endonuclease revealed a single species of RNA (170 bases) that was protected by the solution probe. DDT1 MF-2 cells were found to contain 0.38 pg of .beta.AR mRNA per .mu.g of total cellular RNA. Incubation (16 hr) with isoproterenol decreased .beta.AR mRNA levels in cells by 40%. This agonist-induced decrease in receptor mRNA levels was found to be dependent on the time of incubation and the dose of agonist. The decrease in .beta.AR mRNA was half-maximal at 0.1-0.5 .mu.M isoproterenol. The .beta.-adrenergic antagonists CGP 20712A (.beta.1-selective) and ICI 118,551 (.beta.2-selective) blocked in a dose-dependent fashion the ability of isoproterenol to effect receptor mRNA levels. The .beta.2-adrenergic antagonist displayed a potency 25-fold greater than that of the .beta.1-adrenergic antagonist, in agreement with the subtype of receptor (.beta.2) expressed by these cells. For down-regulated cells in which receptor mRNA levels declined in response to agonist, the addition of the antagonist ligand (-)-propranolol (1 .mu.M) was able to restore receptor mRNA levels to 90% of the control value within 12 hr. Full recovery of steady-state .beta.AR mRNA was achieved within 60 hr. These studies provide a molecular explanation for the down-regulation of GTP-binding regulatory protein (G protein)-linked cell-surface receptors that accompanies desensitization.