Investigation of the extracellular accessibility of the connecting loop between membrane domains I and II of the bradykinin B2 receptor

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane spanning alpha-helices. To analyze the topology of the bradykinin B-2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. We found that a segment with predicted intracellular orientation according to the rhodopsin model, the connecting loop between membrane domains I and II of the bradykinin B-2 receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B-2 receptors, Extracellular orientation of this loop was further confirmed by the substituted cysteine accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modification by a membrane impermeant maleimide, In addition, this segment seemed to be involved in B-2 receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B-2 receptor activation, and (ii) site-directed antibodies to the connecting loop between membrane domains I and II were agonists, The agonistic activity of the antibodies was suppressed by the B-2 antagonist HOE140 confirming the B-2 specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B-2 receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.