Investigation of the extracellular accessibility of the connecting loop between membrane domains I and II of the bradykinin B-2 receptor

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane spanning a lpha-helices. To analyze the topology of the bradykinin B-2 receptor, we ra ised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. We found that a segm ent with predicted intracellular orientation according to the rhodopsin mod el, the connecting loop between membrane domains I and II of the bradykinin B-2 receptor, was accessible to site-directed antibodies on intact fibrobl asts, A431 cells, or COS cells expressing human B-2 receptors, Extracellula r orientation of this loop was further confirmed by the substituted cystein e accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modificat ion by a membrane impermeant maleimide, In addition, this segment seemed to be involved in B-2 receptor activation, since (i) thiol modification of cy steine 94 partially suppressed B-2 receptor activation, and (ii) site-direc ted antibodies to the connecting loop between membrane domains I and II wer e agonists, The agonistic activity of the antibodies was suppressed by the B-2 antagonist HOE140 confirming the B-2 specificity of the antibody-genera ted signal. The extracellular orientation of the connecting loop between me mbrane domains I and II suggests a topology of the B-2 receptor different f rom rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.