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

AUTOR(ES)

Hadcock, J R

RESUMO

Incubation of DDT1 MF-2 hamster vas deferens cells with beta-adrenergic agonists results in a time- and concentration-dependent decreases 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 microgram 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 microM 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 microM) 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.

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