Novel pharmacology and distinct protein interactions of human alpha(1)-adrenergic receptor subtypes.

摘要

Norepinephrine and epinephrine play important roles in a variety of physiological functions through adrenergic receptors (ARs). Three ARs subfamilies (alpha 1, alpha2 beta), each containing three subtypes (alpha 1A, alpha1B, alpha1D, alpha2A, alpha 2B, alpha2C, beta1, beta2, beta 3), are G protein coupled receptors. Three subtypes in each family couple with distinct G protein signaling, but show slight differences in pharmacology and function. Therefore, understanding the differences among subtypes will benefit development of drugs specifically targeting diseases caused by particular subtypes.; The results present in this dissertation provide three new approaches to differentiate three alpha1-AR subtypes: (1) new tools: conopeptide rho-TIA was characterized as a 10-fold selective non-competitive inhibitor at the alpha1B-AR but a competitive inhibitor at alpha 1A- and alpha1D-ARs in radioligand binding assays. Receptor N-termini were not involved in this peptide-receptor interaction because receptor constructs without the N-terminus showed similar binding affinities to 125I-BE as the full-length receptors. Alanine-scanning mutagenesis of rho-TIA showed some mutations, such as F18A, could yield higher subtype-selectivity and may serve as novel templates for design of highly alpha1B-AR-selective drugs; (2) receptor C-termini involved in subtype-specific regulation of receptor functions and internalization: alpha1-AR C-termini are not involved in receptor expression, pharmacology or oligomerization. However, alpha1-AR C-terminally truncated and chimeric mutants showed subtype-specific effects on Galphaq/11/Ca2+ signaling. The alpha1A-AR C-terminus plays a positive effect on receptor signaling, while that of the alpha1B-AR negatively regulates Ca2+ mobilization. The alpha1B-AR C-terminus, but not that of the alpha1A-AR, is required for receptor internalization; (3) new subtype-specific binding partners: alpha-syntrophin, a PDZ-domain-containing protein, was found to be an alpha1D-AR selective binding partner in yeast two-hybrid screening. The PDZ domains of alpha, beta1, and beta2-syntrophins were shown to specifically interact with the alpha1D-AR C-terminus but not with those of alpha1A- and alpha1B-ARs in fusion protein blot overlay assays. Mutation of the receptor C-terminal PDZ-interacting motif drastically impaired receptor stability and receptor-mediated Galpha q/11 signaling, while overexpression of alpha-syntrophin improved receptor expression, suggesting that syntrophins modulate receptor functions through a PDZ-domain-mediated interaction.