Modeling and mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of transmembrane helix V...

A 3-dimensional model of the seven transmembrane helical segments (TMs) of the human alpha 1a-adrenoceptor was initially built by analogy to the known structure of bacteriorhodopsin. However, the rotational orientation of TM V about its helical axis, and the roles of several TM V residues in ligand binding and receptor activation remained in question. Accordingly, we determined the effects of six site-specific mutations in TM V on binding affinity and functional potency of a structurally diverse series of agonists and antagonists. Mutation of Ser 192 and Phe 193 disrupted the binding of many of the tested ligands, as measured by displacement of [3H]prazosin. In addition, mutation of Ser 188, Ser 192, and Phe 193 disrupted receptor activation, as measured by [3H]inositol phosphate formation. On the basis of these results, a specific rotational orientation of TM V is proposed as part of a revised receptor model, which also takes into account more recently reported information about the structure of rhodopsin. This revised alpha 1a-adrenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of norepinephrine, respectively, in the G-protein coupled receptor state.