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 (Y2) receptor. Although ligand binding and GTPγS binding studies supported high functional Y2 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 Y2 receptor in SMS-KAN cells, the transient calcium (Ca2+) mobilization assay in the fluorimetric imaging plate reader (FLIPR) format was established by stably expressing a chimeric G protein Gqi9. This manipulation resulted in robust mobilization of Ca2+ 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 Y2 receptor. The selective Y2 antagonist BIIE0246 competitively inhibited NPY-mediated Ca2+ transients in SMS-KAN/Gqi9 cells with a pA2 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.