Structure-function relationship of different domains of the rat corticotropin-releasing factor receptor

  • Sydow S
  • Radulovic J
  • Dautzenberg F
 et al. 
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Abstract

The significance of different domains of corticotropin-releasing factor receptor, type 1, (CRFR1) for ligand binding and cAMP accumulation was investigated with C-terminally truncated forms of rat CRFR1 (rCRFR1) tagged by a sequence of six histidine residues (His-tag) to facilitate protein purification and identification. These different forms of the receptor were N-glycosylated and transported properly to the membranes of transfected mammalian cells as indicated by Western blot analysis and immunocytochemical staining with two polyclonal antibodies developed against the N- and C-terminus of rCRFR1. The N-terminal fragment, rCRFR1(23-121), expressed in Escherichia coli bound oCRF specifically, but with low affinity. Several mutants lacking transmembrane domain (TM) 7 and the C-terminus exhibited similarly low affinities to oCRF after expression in transfected mammalian cells. None of these cells produced significant amounts of cAMP after exposure to oCRF. Only mutants containing the N-terminus, all loops and TMs bound oCRF and produced cAMP with high affinity (K(d) = 62 nM) and efficacy (EC50= 0.8 nM). The additional presence of the C-terminus provided similar characteristics (K(d) = 5 nM, EC50= 0.3 nM) as known for the native receptor. It is suggested on the basis of these data that the last extracellular loop is involved in ligand binding.

Author-supplied keywords

  • CHO-K1 cell
  • CRF binding
  • CRFR1 antibodies
  • Corticotropin-releasing factor receptor
  • Cyclic AMP
  • HEK293 cell
  • Histidine tag
  • N-glycosylation

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