Agonist-dependent immobilization of chimeric bombesin/GRP receptors: Dependence on c-Src activity and dissociation from internalization

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a de creased mobile fraction compared to a freely mobile plasma membrane protein . Recently, interest has focused on proteins other than heterotrimeric G-pr oteins that interact with GPCRs as scaffolding structures that affect recep tor signal transduction. In order to investigate the physical state of rece ptors before and after agonist, we used fluorescence recovery after photobl eaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to t he intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) e xpressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells a t 37 degreesC, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was indep endent of endocytosis, Concurrently, the remaining mobile GFP-GRP receptors showed an increase in the diffusion rate with the halftime of fluorescent recovery, tau (1/2) = 46 +/- 7 s for untreated cells, decreasing to tau (1/ 2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-sp ecific inhibitor PP-2 (10 muM) blocked GFP-GRP receptor immobilization whil e treatment with the inactive analog PP-3 (10 muM) did not affect receptor immobilization, These data suggest that agonist-bound GPCR have increased p lasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity. (C) 2001 Aca demic Press.