Structure-function relationship of different domains of the rat corticotropin-releasing factor receptor.
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 (Kd = 62 nM) and efficacy (EC50 = 0.8 nM). The additional presence of the C-terminus provided similar characteristics (Kd = 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.
Sydow
S
S
Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Göttingen, Germany. sydow@mail.mpiem.gwdg.de
Radulovic
J
J
Dautzenberg
F M
FM
Spiess
J
J
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
D010446:Peptide Fragments; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D003346:Corticotropin-Releasing Hormone