Alanine Scanning Mutagenesis of the Second Extracellular Loop of Type 1 Corticotropin-Releasing Factor Receptor Revealed Residues Critical for Peptide Binding

Abstract

Upon binding of the corticotropin-releasing factor (CRF) analog sauvagine to the type 1 CRF receptor (CRF 1 ), the amino-terminal portion of the peptide has been shown to lie near Lys257 in the receptor's second extracellular loop (EL2). To test the hypothesis that EL2 residues play a role in the binding of sauvagine to CRF 1 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 CRF 1 . 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 CRF 1 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 CRF 1 . This is the first time that a specific region of CRF 1 has been implicated in detailed interactions between the receptor and the amino-terminal portion of peptides belonging to the CRF family.

Structural-Functional Analysis of the Third Transmembrane Domain of the Corticotropin-releasing Factor Type 1 Receptor

“…3C). Astressin is a CRF peptide antagonist that is less sensitive to receptor activation-associated conformational changes than agonists, and it interacts with the extracellular N-domain of CRF 1 R (8,15 (Fig. 4A), in a manner similar to the effect of the MTSEA reaction that adds a Lys-like side chain to F203…”

“…After 1-5 days, frozen lysates were thawed and centrifuged at 1800 ϫ g for 10 min at 4°C, and the supernatants were neutralized with 2 N NaOH. Quantification of cAMP in the neutralized supernatants was performed using a competitive binding assay as described previously (15). In brief, supernatants were transferred to polypropylene mini-tubes (20 l/tube) containing buffer A (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM EDTA) with 1-1.5 nM [2,8-3 H]cAMP (PerkinElmer Life Sciences).…”

“…The amount of membrane used was adjusted to ensure that the specific binding was always equal to or less than 10% of the total concentration of the added radioligand. -sauvagine binding were determined from heterologous and homologous competition data, respectively, as described previously using Prism 4.0 (15).…”

“…The homogenates were centrifuged at 16,000 ϫ g for 10 min at 4°C, and the membrane pellets were resuspended in 1 ml of buffer B (buffer H containing 0.1% bovine serum albumin, pH 7.2, at 20°C). The membrane suspensions were diluted in buffer B and used for homologous or heterologous competition binding studies as described previously (15). In brief, aliquots of diluted membrane suspensions (50 l) were added into low retention tubes (Kisker-Biotech, Steinfurt, Germany), containing buffer B and 20 -50 pM 125 I-Tyr 0 -sauvagine with or without increasing concentrations of Tyr 0 -sauvagine (homologous competition binding), sauvagine, astressin, or antalarmin (heterologous competition binding) (American Peptide Co., Sunnyvale, CA).…”

The “homeostasis hormone” and its CRF1 receptor. From structure to function

“…94,95 The latter finding is in agreement with the results of an alanine mutagenesis study, which has suggested that Trp259 and Phe260 in the EL2 of CRF1 receptor most likely interact with ligands, and specifically with the amino-terminal residues 8-10 of SVG and the corresponding ones of CRF. 96 Furthermore, this study has proposed that the interaction between the amino-terminal region of CRF family peptides and Trp259 and Phe260 of CRF1 seems to be critical for receptor activation and the subsequent appearance of a biological effect. 96 In addition to EL2, the first extracellular loop (EL1) of CRF1 has been demonstrated as playing a part in peptide binding, given that the amino-terminally located residues 17 and 22 of UCN analogs have been shown in a recent study to be located in close proximity to residues Trp170-Glu179 in the EL1 of CRF1.…”