Abstract

Family B of G protein-coupled receptors (GPCRs) is composed of receptors that bind peptides, such as secretin, glucagon, parathyroid hormone, and corticotropin releasing factor (CRF), which play critical physiological roles. These receptors, like all GPCRs, share a common structural motif of seven membrane-spanning segments, which have been proposed to bind small ligands, such as antalarmin, a nonpeptide antagonist of the type 1 receptor for CRF (CRF₁). This leads to the hypothesis that as for family A GPCRs, the binding sites of small ligands for family B GPCRs are on the surface of a water-accessible crevice, the binding-site crevice, which is formed by the membrane-spanning segments and extends from the extracellular surface of the receptor into the plane of the membrane. To test this hypothesis we have begun to obtain structural information about family B GPCRs, using as a prototype the CRF₁, by determining the ability of sulfhydryl-specific methanethiosulfonate derivatives, such as the methanethiosulfonate-ethylammonium (MTSEA), to react with CRF₁ and thus irreversibly inhibit ¹²⁵I-Tyr⁰-sauvagine binding. We found that MTSEA inhibited ¹²⁵I-Tyr⁰-sauvagine binding to CRF₁ and that antalarmin protected against this irreversible inhibition. To identify the susceptible cysteine(s), we mutated, one at a time, four endogenous cysteines to serine. Mutation to serine of Cys211, Cys233, or Cys364 decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. Thus, Cys211, Cys233, and Cys364 at the cytoplasmic ends of the third, fourth, and seventh membrane-spanning segments, respectively, are exposed in the binding site crevice of CRF₁.