Molecular Endocrinology, Vol 7, 85-93, Copyright © 1993 by Endocrine Society

ARTICLES

Mutation of a highly conserved acidic residue present in the second intracellular loop of G-protein-coupled receptors does not impair hormone binding or signal transduction of the luteinizing hormone/chorionic gonadotropin receptor

Z Wang, H Wang and M Ascoli
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242-1109.

Sequence alignment shows that there is a highly conserved acidic residue (D or E) at the boundary between the third transmembrane domain and the second intracellular loop of the superfamily of G-protein- coupled receptors. Previous mutagenesis studies demonstrated that substitution of this acidic residue in the beta 2-adrenergic, muscarinic m1, and alpha 2A-adrenergic receptors by the corresponding amide preserved high affinity agonist binding, but significantly reduced or completely abolished activation of the respective effector. To determine whether the corresponding amino acid residue (E441) played a similar role in the functions of the rat LH/CG receptor, we used site- directed mutagenesis to substitute it by D or Q. The wild-type and mutant receptors (E441D or E441Q) were then transfected into human embryonic kidney 293 cells and tested for their ability to bind hCG and respond to it with increased cAMP accumulation. As predicted, the mutant LH/CG receptors were found to bind hCG with high affinity. In contrast to the results summarized above, however, an E441Q or an E441D mutation in the LH/CG receptor results in only a slight increase in the EC50 for cAMP accumulation without decreasing the maximal response attained. The most remarkable effect of these mutations was on localization of the receptor. Thus, while most of the receptors expressed in cells transfected with the E441D mutant could be detected by measuring hormone binding to intact cells, most of the receptors expressed in cells transfected with the E441Q mutant could be detected only upon solubilization of the cells with detergent.

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