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The subfamily B of G-protein-coupled receptors (GPCRs) consists of receptors that bind peptides, such as secretin, vasoactive intestinal peptide, glucagon, corticotropin-releasing hormone (CRF), which play a fundamental role in body function. These receptors, like all GPCRs, are plasma membrane proteins sharing a common structural motif of seven membrane-spanning domains (TMs), which have been shown to bind small molecules, such as antalarmin, a non-peptide antagonist of the type 1 receptor for CRF (CRF1). This leads to the hypothesis that similar to family A, rhodopsin-like, GPCRs, the TMs of subfamily B GPCRs form a water-accessible crevice, the binding-site crevice, which extends from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed not only by residues that can contact small molecules but also by residues that may play a structural role and affect binding indirectly. However, the lack of considerable structural informat ...
The subfamily B of G-protein-coupled receptors (GPCRs) consists of receptors that bind peptides, such as secretin, vasoactive intestinal peptide, glucagon, corticotropin-releasing hormone (CRF), which play a fundamental role in body function. These receptors, like all GPCRs, are plasma membrane proteins sharing a common structural motif of seven membrane-spanning domains (TMs), which have been shown to bind small molecules, such as antalarmin, a non-peptide antagonist of the type 1 receptor for CRF (CRF1). This leads to the hypothesis that similar to family A, rhodopsin-like, GPCRs, the TMs of subfamily B GPCRs form a water-accessible crevice, the binding-site crevice, which extends from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed not only by residues that can contact small molecules but also by residues that may play a structural role and affect binding indirectly. However, the lack of considerable structural information for the family B GPCRs precludes the support of this hypothesis. To test this hypothesis we started obtaining structural information about subfamily B GPCRs, using as prototype the CRF1 and determining its ability to react with the charged, hydrophilic, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) derivative, MTSethylammonium (MTSEA). The reaction of MTSEA with CRF1 was tested by its ability to irreversibly inhibit [125I]-sauvagine binding to receptor. Reaction of MTSEA with CRF1 inhibited [125I]-sauvagine binding to CRF1. These results, in conjunction with that the antagonist antalarmin protected against irreversible inhibition by the MTSEA, suggest that MTSEA reacted with the sulfhydryl of one or more cysteines of CRF1. To identify the susceptible cysteine(s), we mutated to serine, one at a time, the four endogenous cysteine residues, Cys128, Cys211, Cys233 and Cys364, which are located in the first (TM1), third (TM3), fourth (TM4) and seventh (TM7) membrane spanning segment of CRF1. In contrast to Cys128 mutation, substitution of Cys211, Cys233 and Cys364 by serine decreased the susceptibility of sauvagine binding to irreversible inhibition by MTSEA. These results suggest that Cys211, Cys233 and Cys364 are exposed in the binding-site crevice of CRF1, and their reaction with MTSEA decreased sauvagine binding to receptor. Subsequently we mutated, one at a time, 18 residues (engineered Cys) of the third membrane spanning segment (TM3) of CRF1 to cysteine, and tested the accessibilities of the engineered Cys for reaction with the MTSEA. Six of the mutant receptors reacted with MTSEA, suggesting that the side chains of these residues are exposed in the binding-site crevice, of CRF1. The pattern of accessibility was consistent with an alpha-helical conformation. In contrast to membrane spanning segments of CRF1 which have been proposed to bind the small non-peptide antagonists, the extracellular amino-terminal regions and the three extracellular loops of CRF1 interact with the larger peptides belonging to CRF family, such as CRF and sauvagine. In specific previous studies have been shown that upon binding of sauvagine to CRF1, the amino-terminal portion of the peptide lies near Lys257 in the receptor?s second XI extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF1 we carried out an alanine-scanning mutagenesis study to determine the functional role of EL2 residues (Leu251 to Val266). Only the W259A, F260A, and W259A/F260A mutations reduced the binding affinity and potency of sauvagine. In contrast, these mutations did not seem to significantly alter the overall receptor conformation, in that they left unchanged the affinities of the ligands astressin and antalarmin that have been suggested to bind to different regions of CRF1. The W259A, F260A, and W259A/ F260A mutations also decreased the affinity of the endogenous ligand, CRF, implying that these residues may play a common important role in the binding of different peptides belonging to CRF family. Parallel amino acid deletions of the two peptides produced ligands with various affinities for wild-type CRF1 compared with the W259A, F260A, and W259A/F260A mutants, supporting the interaction between the amino-terminal residues 8 to 10 of sauvagine and the corresponding region in CRF with EL2 of CRF1. This is the first time that a specific region of CRF1 has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family
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