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) 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.
Assil-Kishawi
Iman
I
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Abou-Samra
Abdul B
AB
D029845:Amphibian Proteins; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D036361:Peptide Hormones; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism*; D014665:Vasodilator Agents / Q000378:metabolism; D000099052:CRF Receptor, Type 1
D029845:Amphibian Proteins; D003432:Cross-Linking Reagents; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D014665:Vasodilator Agents; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D001120:Arginine; D000242:Cyclic AMP; D008239:Lysine; D003488:Cyanogen Bromide