Extracellular cysteines of the corticotropin-releasing factor receptor are critical for ligand interaction.
The corticotropin-releasing factor receptor (CRF-R) contains six conserved cysteines in its amino-terminal domain (C30, C44, C54, C68, C87, and C102) and one cysteine in its first and second extracellular loops (C188 and C258, respectively). Additionally, several other cysteines are located in the transmembrane domains (C128, C211, C233, and C364) and first intracellular loop (C150). Reduction of disulfide bonds with DTT decreased CRF binding to detergent-solubilized membranes, suggesting an important role for disulfide bonds in ligand recognition. Therefore, site-directed mutagenesis was used to introduce single and paired Cys (C) to Ser (S) or Ala (A) mutations. A silent nine amino acid tag from c myc was introduced in the amino terminus of the mouse CRF-R. With the exception of C258S and C188S/C258S mutations, all C to S or to A receptor mutants had good surface expression that was at least 52.5% of control. C30S, C54S, and C30S/C54S mutations had good CRF binding and CRF-stimulated cAMP accumulation. No CRF binding was detected for the C44S, C68S, C87S, C102S, C188S, C258S, C30S/C44S, C30S/C68S, C54S/C68S, C87S/C102S, and C188S/C258S mutants, while CRF-stimulated cAMP accumulation occurred with high EC50 values. In particular, receptors carrying double mutations, C44S/C102S and C68S/C87S, had an improved signaling property as compared to receptors carrying the respective single cysteine mutations. These data, together with the effects of DTT on CRF binding, indicate that disulfide bridges are important for receptor functions. Functional data from single and paired cysteine mutations suggest potential pairings between C44 and C102, C68 and C87, and C188 and C258 that are critical for ligand-receptor interactions.
Qi
L J
LJ
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Leung
A T
AT
Xiong
Y
Y
Marx
K A
KA
Abou-Samra
A B
AB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D018019:Receptors, Corticotropin-Releasing Hormone; D003346:Corticotropin-Releasing Hormone; D003545:Cysteine