Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain.
OBJECTIVE
Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands.


METHODS
We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays.


RESULTS
Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did.


CONCLUSIONS
Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.
Beyermann
M
M
Leibniz-Institut für Molekulare Pharmakologie im FV Berlin e.V., Berlin, Germany. beyermann@fmp-berlin.de
Heinrich
N
N
Fechner
K
K
Furkert
J
J
Zhang
W
W
Kraetke
O
O
Bienert
M
M
Berger
H
H
D029845:Amphibian Proteins; D002460:Cell Line; D002462:Cell Membrane; D003346:Corticotropin-Releasing Hormone / Q000819:agonists* / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006801:Humans; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides; D011485:Protein Binding; D011487:Protein Conformation; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D054832:Urocortins; D000099052:CRF Receptor, Type 1
D029845:Amphibian Proteins; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs