Use of constitutive G protein-coupled receptor activity for drug discovery

This article describes the behavior of transiently transfected human recept ors into melanophores and the potential use of constitutive receptor activi ty to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably lea ds to increased levels of receptor expression. This leads to an increased r esponse to agonists (both maxima and potency) and, in some cases, an agonis t-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated recep tor to cause elevated basal cellular responses. This was observed as a decr ease in the transmittance of light through melanophores (consistent with G( s) protein activation) and increased response to human calcitonin. The rece ptor-mediated nature of this response was confirmed by its reversal with th e hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 ei ther increased or decreased constitutive hCTR2 activity, suggesting that th e constitutive system was a sensitive discriminator of positive and negativ e ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent wi th G(i)-protein activation). NPY1 cDNA produced little constitutive respons e on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potenc y) with increasing cDNA transfection. The receptor/G(i)-protein nature of b oth the constitutive and agonist-mediated responses was confirmed by elimin ation with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applic ability of this approach for the general screening of G protein-coupled orp han receptors.