MODELING AND MUTAGENESIS OF THE HUMAN ALPHA(1A)-ADRENOCEPTOR - ORIENTATION AND FUNCTION OF TRANSMEMBRANE HELIX-V SIDE-CHAINS

A 3-dimensional model of the seven transmembrane helical segments (TMs ) of the human alpha(1a)-adrenoceptor was initially built by analogy t o the known structure of bacteriorhodopsin. However, the rotational or ientation of TM V about its helical axis? and the roles of several TM V residues in ligand binding and receptor activation remained in quest ion. Accordingly, we determined the effects of six site-specific mutat ions in TM V on binding affinity and functional potency of a structura lly diverse series of agonists and antagonists. Mutation of Ser 192 an d Phe 193 disrupted the binding of many of the tested ligands, as meas ured by displacement of [H-3]prazosin. In addition, mutation of Ser 18 8, Ser 192, and Phe 193 disrupted receptor activation, as measured by [H-3]inositol phosphate formation. On the basis of these results, a sp ecific 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)-a drenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of n orepinephrine, respectively, in the G-protein coupled receptor state.