J. Biol. Chem., Vol. 262, Issue 34, 16439-16443, Dec, 1987

Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity [published erratum appears in J Biol Chem 1988 Feb 25;263(6):3050]

CD Strader, RA Dixon, AH Cheung, MR Candelore, AD Blake and IS Sigal
Department of Biochemistry and Molecular Biology, Merck Sharp, and Dohme Research Laboratories, Rahway, New Jersey 07065.

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258- 270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.
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