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

Abstract

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radio-ligands [Tyr0,Gln1]SVG (125I-YQS) and [Tyr0,Gln1, Leu17]SVG (125I-YQLS) were examined. 125I-YQLS or 125I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the ε amino groups of lysine residues that have a molecular distance of 11.4 Å. DSS specifically and efficiently cross-linked 125I-YQLS and 125I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys110, Lys111, Lys113, Lys257, and Lys262) that can cross-link to the 4 lysine residues (Lys16, Lys22, Lys25, and Lys27) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to 125I-YQLS or 125I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys16 of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys257 in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys257 of CRFR1 lies in close proximity (11.4 Å) to Lys16 of SVG.