AMINO-TERMINAL POLYMORPHISMS OF THE HUMAN BETA(2)-ADRENERGIC RECEPTOR IMPART DISTINCT AGONIST-PROMOTED REGULATORY PROPERTIES

We have recently delineated three naturally occurring polymorphisms of the human beta(2)-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg 16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to G(s), resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 mu M isoproterenol, wild-type beta(2)AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly + Gln27-->Glu also underwent an increased downregulation compared to wild-type beta(2)AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNB. minimally increased during agonist exposure, and wild-type beta(2)AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein. On Western blots, the Gln27-->Glu receptor did not appear to be expressed in the fully mature form as compared to wild-type beta(2)AR, suggesting that conformational alterations of this receptor lead to depressed receptor degradation during agonist exposure. These results indicate that common polymorphisms of the beta(2)AR at the N-terminus impart distinct phenotypes of agonist-promoted regulation and may explain the observed heterogeneity of in vivo responsiveness and regulation of beta(2)AR.