CRF-R

CRF receptors (type 1, CRF1, and type 2, CRF2) belong to subfamily B of G-protein coupled receptors (GPCRs). The larger peptides (corticotropin releasing factor, CRF, and CRF-like peptides) bind to the extracellular domains of CRF receptors whereas the smaller non-peptide antagonists have been proposed to contact residues of the membrane-spanning domains of receptors. In contrast to family A GPCRs, little is known about the structure of CRF receptors, as well as for all receptors belonging to family B GPCRs. The efforts in our laboratory aim 1) to obtain structural information for CRF receptors, by applying the substituted cysteine accessibility method (SCAM) and 2) to determine the role of the amino acids of CRF receptors in the binding of CRF and CRF-like peptides, natural and synthesized in the laboratory of our collaborator Dr. Theodoros Tselios (University of Patras) and Dr. Vassiliki Magafa (University of Patras). We recently found that two amino acids in the second extracellular loop of the type 1 CRF receptor (CRF1) play an important role in peptide binding, by interacting with their aminoterminal amino acids. In contrast to the extracellular regions of CRF1, which are involved in the binding of peptides, the membrane-spanning segments of receptor have been proposed to bind the small non-peptide antagonists. This led to the hypothesis that as for family A GPCRs, the binding sites of small ligands for the CRF1 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 obtained structural information about CRF1, by determining the ability of sulfhydryl-specific methanethiosulfonate derivatives, such as the methanethiosulfonate-ethylammonium (MTSEA), to react with CRF1 and thus irreversibly inhibit radioligand binding. We found that MTSEA inhibited radioligand binding to CRF1 by reacting with Cys211, Cys233, and Cys364 at the cytoplasmic ends of the third, fourth, and seventh membrane-spanning segments of CRF1, suggesting that these residues are exposed in the binding site crevice of receptor. We are now characterizing the residues lining the binding-site crevice of CRF1 by applying SCAM.