J Biol Chem, Vol. 274, Issue 21, 14773-14778, May 21, 1999

Investigation of the Extracellular Accessibility of the Connecting Loop between Membrane Domains I and II of the Bradykinin B₂ Receptor

Ursula Quitterer, Essam Zaki^§, and Said AbdAlla^§

From the  Institut für Pharmakologie und Toxikologie der Universität, Versbacher Straße 9, 97078 Würzburg, Germany and the ^§ Genetics Engineering and Biotechnology Research Institute (GEBRI), Alexandria, Egypt

In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning -helices. To analyze the topology of the bradykinin B₂ receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and car- boxyl 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 B₂ receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B₂ 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 B₂ receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B₂ 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 B₂ antagonist HOE140 confirming the B₂ specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B₂ receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.