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Vol. 57, Issue 1, 125-134, January 2000
Departments of Receptor Biochemistry (G.C., C.W., K.Q., C.K.J.,
T.K.) and of Molecular Sciences (J.W., S.A., W.-J.C.), Glaxo Wellcome
Research and Development, Research Triangle Park, North Carolina
This article describes the behavior of transiently transfected human
receptors into melanophores and the potential use of constitutive
receptor activity to screen for new drug entities. Specifically,
transient transfection of melanophores with different concentrations of
receptor cDNA presumably leads to increased levels of receptor
expression. This leads to an increased response to agonists (both
maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the Gs protein-coupled human calcitonin
receptor type 2 (hCTR2) cDNA produced sufficient levels of
constitutively activated receptor to cause elevated basal cellular
responses. This was observed as a decrease in the transmittance of
light through melanophores (consistent with Gs protein
activation) and increased response to human calcitonin. The
receptor-mediated nature of this response was confirmed by its reversal
with the hCTR2 peptide inverse agonist AC512. A collection of ligands
for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained
with Gi-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with
Gi-protein activation). NPY1 cDNA produced little
constitutive response on transfection, whereas maximal levels of
constitutive activity ranging from 30 to 45% were observed for the
other Gi-protein-coupled receptors. Responses to agonists
for these receptors increased (both maxima and potency) with increasing
cDNA transfection. The receptor/Gi-protein nature of both
the constitutive and agonist-mediated responses was confirmed by
elimination with pertussis toxin pretreatment. These data are discussed
in terms of the theoretical aspects of constitutive receptor activity
and the applicability of this approach for the general screening of
G protein-coupled orphan receptors.
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