Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.
OBJECTIVE
According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different.


METHODS
We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay.


RESULTS
The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism.


CONCLUSIONS
The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).
Berger
H
H
Department of Peptide Chemistry/Biochemistry, Leibniz Institute of Molecular Pharmacology, Berlin, Germany.
Heinrich
N
N
Wietfeld
D
D
Bienert
M
M
Beyermann
M
M
D000494:Allosteric Regulation; D029845:Amphibian Proteins; D001667:Binding, Competitive; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000494:pharmacology; D036361:Peptide Hormones; D010455:Peptides / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D054832:Urocortins; D000099052:CRF Receptor, Type 1
D029845:Amphibian Proteins; D006727:Hormone Antagonists; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D011743:Pyrimidines; D011758:Pyrroles; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; C103016:antalarmin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000099052:CRF Receptor, Type 1; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; C045089:corticotropin releasing hormone (9-41); D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs