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 decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, 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 independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time 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-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.
Young
S H
SH
Division of Digestive Diseases, University of California, Los Angeles, California 90095, USA.
Walsh
J H
JH
Rozengurt
E
E
Slice
L W
LW
D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008560:Membrane Fluidity; D015688:Oncogene Protein pp60(v-src) / Q000037:antagonists & inhibitors; D021381:Protein Transport; D018004:Receptors, Bombesin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D008164:Luminescent Proteins; D018004:Receptors, Bombesin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015688:Oncogene Protein pp60(v-src); D019061:src-Family Kinases