Max Planck Scientists Report Molecular Details On The Interaction Of The Stress Hormone Corticotropin-Releasing Factor (CRF) And Its Receptor

May 06, 1998

Corticotropin-releasing factor (CRF) is a 41 residue polypeptide hormone that is recognized as an early chemical signal in the response to stress. The hormone exhibits its actions through plasma membrane receptors which have been characterized from several sources. The molecular interaction between these receptors and CRF is under investigation by several laboratories. It is expected that further molecular information on the receptor will enhance the CRF biology and thereby stress research.

One year ago, scientists of the Department of Molecular Neuroendocrinology of the Max Planck Institute for Experimental Medicine in Goettingen/ Germany published the first report on the predicted amino acid sequences of two frog (Xenopus laevis) CRF receptors (Frank M. Dautzenberg, Konstanze Dietrich, Monika R. Palchaudhuri and Joachim Spiess. Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus Laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor. Journal of Neurochemistry pp.1640-1649, 1997). Principal investigator of the project was Dr. Frank Dautzenberg. The group found that frog - like all other species investigated - synthesizes two receptor types (types 1 and 2). Frog CRF receptor 1 (CRF-R1) exhibits a surprisingly high ligand selectivity. Human/rat CRF or urocortin are bound with significantly higher affinity than ovine CRF or sauvagine, a frog-specific CRF analog. These results contrast with the observation confirmed by many groups that mammalian CRF-R1 does not discriminate between the naturally occurring CRF analogs mentioned above.

On the basis of this difference, it was possible to identify the ligand-specific domain of frog CRF-R1 and to determine whether mammalian CRF-R1 could be rendered ligand-selective by introduction of amino acids recognized to be crucial for the ligand-specificity of frog CRF-R1. The strategy and results of these experiments have very recently been published in the Proceedings of the National Academy of Sciences (Frank M. Dautzenberg, Sandra Wille, Ragna Lohmann and Joachim Spiess. Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: Implications for the ligand binding site. Proc.Nat.Acad.Sci., Vol 95 (9), April 28, 1998).

The objective of this project was approached by generating cDNA constructs coding for chimeric molecules which consisted N-terminally of frog CRF-R1 and C-terminally of human CRF-R1 or vice versa. These constructs were transfected into human embryonic kidney (HEK) cells. The biological activity was tested by monitoring the production of the second messenger cAMP by the transfected cells in response to increasing concentrations of various CRF analogs. The affinity of various ligands was determined by the analysis of binding data obtained from experiments with membranes of the transfected cells.

With this approach, the ligand-selective region was mapped to the amino acid stretch from residue 70 to residue 89 of frog CRF-R1. By site-directed mutagenesis, it was found that respective replacement of glutamine-76, glycine-81, valine-83, histidine-88 and leucine-89 of frog CRF-R1 by arginine-76, asparagine-81, glycine-83, leucine-88 and alanine-89 of human CRF-R1 abolished the ligand-selectivity of the frog receptor. On the other side, the reverse procedure produced a human receptor with the ligand-selectivity of frog CRF-R1.

From these results it was concluded that the identified region is part of the ligand-binding site of human and frog CRF-R1 and that human and frog CRF-R1 may derive from a common ancestor molecule.
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Max-Planck-Gesellschaft

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