Sauvagine cross-links to the second extracellular loop of the corticotropin-releasing factor type 1 receptor

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radio-ligands [Tyr(0),Gln(1)]SVG (I-125-YQS) and [Tyr(0),Gln(1), Leu(17)] SVG (I-125-YQLS) were examined. I-125-YQLS or I-125-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 Angstrom. DSS specifically and efficiently cross-linked I-125-YQLS and I-125-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 I-125-YQLS or I-125-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 Angstrom) to Lys(16) of SVG.