Stimulation of neuropeptide Y-mediated calcium responses in human SMS-KAN neuroblastoma cells endogenously expressing Y2 receptors by co-expression of chimeric G proteins.
Human SMS-KAN neuroblastoma cells endogenously express the neuropeptide Y (NPY) type 2 (Y(2)) receptor. Although ligand binding and GTPgammaS binding studies supported high functional Y(2) receptor expression, only weak coupling to the natural second messenger cyclic AMP was observed. The main reason was the low responsiveness of SMS-KAN cells to forskolin, a direct activator of adenylyl cyclases. In order to obtain a cell-based functional assay for the Y(2) receptor in SMS-KAN cells, the transient calcium (Ca(2+)) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gq(i9). This manipulation resulted in robust mobilization of Ca(2+) after challenge with various NPY-related agonists in a 384-well format. The sensitivity of the FLIPR readout was in the low nanomolar range for NPY agonists and comparable to that of the recombinant Y(2) receptor. The selective Y(2) antagonist BIIE0246 competitively inhibited NPY-mediated Ca(2+) transients in SMS-KAN/Gq(i9) cells with a pA(2) value of 7.39+/-0.1. This is the first evidence that an endogenously expressed G protein-coupled receptor couples to an overexpressed chimeric G protein, thereby functionally responding in the FLIPR readout.
Dautzenberg
Frank M
FM
Axovan Ltd., Gewerbestrasse 16, 4123 Basel, Switzerland. fdautzen@prdbe.jnj.com <fdautzen@prdbe.jnj.com>
D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009478:Neuropeptide Y / Q000502:physiology*; D017476:Receptors, Neuropeptide Y / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology
D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C095889:neuropeptide Y2 receptor; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium