STRUCTURE-FUNCTION RELATIONSHIP OF DIFFERENT DOMAINS OF THE RAT CORTICOTROPIN-RELEASING FACTOR-RECEPTOR

The significance of different domains of corticotropin-releasing facto r receptor, type I, (CRFR1) for ligand binding and cAMP accumulation w as investigated with C-terminally truncated forms of rat CRFR1 (rCRFR1 ) tagged by a sequence of six histidine residues (His-tag) to facilita te protein purification and identification. These different forms of t he receptor were N-glycosylated and transported properly to the membra nes of transfected mammalian cells as indicated by Western blot analys is and immunocytochemical staining with two polyclonal antibodies deve loped against the N- and C-terminus of rCRFR1. The N-terminal fragment , rCRFR1(23-121), expressed in Escherichia coli bound oCRF specificall y, 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 p roduced significant amounts of cAMP after exposure to oCRF. Only mutan ts containing the N-terminus. all loops and TMs bound oCRF and produce d cAMP with high affinity (K-d = 62 nM) and efficacy (EC50 = 0.8 nM). The additional presence of the C-terminus provided similar characteris tics (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 lo op is involved in ligand binding. (C) 1997 Elsevier Science B.V.