Abstract
Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.
Highlights
Corticotropin-releasing factor (CRF),1 sauvagine (SVG), urocortin-I (UCN-I), and urotensin-I (UTS) bind to the CRF receptors, corticotropin-releasing factor receptor type 1 (CRFR1) and CRFR2, previously characterized from various species
The cross-linked receptor was resolved as a broad band of ϳ80 kDa, which is consistent with the predicted molecular mass of the glycosylated CRFR1 (Fig. 1A)
We reported that the oxidation-resistant sauvagine radioligand, 125I-YQLS, which exhibits high affinity binding to the CRF receptors, cross-links efficiently to mCRFR1 through 1 of the 4 lysine residues of the ligand to 1 of the lysine residues of the receptor clustered at the juxtamembrane region and/or the second extracellular loop [16]
Summary
Materials—Unless indicated, all chemicals were purchased from Sigma Chemicals. Na125I was purchased from PerkinElmer Life Sciences. The peptides, hCRF-(1– 41) (CRF), [Tyr0,Gln1]SVG (YQS), and [Tyr0,Gln1,Leu17]SVG (YQLS) were synthesized in the Massachusetts General Hospital Biopolymer Facility They were HPLC-purified and analyzed by mass spectroscopy, amino-terminal sequencing, and acid hydrolysis. Intact COS-7 cells transiently transfected with mCRFR1 or mCRFR1 mutants were plated into 24-well plates, and a binding assay was performed when cells reached 90 –95% confluency. The cells were rinsed with a Tris-based binding buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 2 mM CaCl2, 5 mM KCl, 5% heat-inactivated horse serum, 0.5% heat-inactivated fetal bovine serum). 125I-YQLS or 125I-YQS (1,000,000 cpm/well) was added in HEPES binding buffer (25 mM HEPES, pH 7.6, 125 mM NaCl, 5 mM KCl, 5% heat-inactivated horse serum, 0.5% heatinactivated fetal bovine serum) for 2– 4 h at room temperature. The cells were rinsed with PBS, lysed with SDS sample buffer, and analyzed on a 5–20% SDS-polyacrylamide gel. The proteins were analyzed on a 5–20% gradient SDS gel or on Tricine/SDS-PAGE (12% acrylamide) according to the method of Schagger and von Jagow [19] and autoradiographed using x-ray film or a phosphorimager screen
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
