EFFECT OF GASTRIN-RELEASING PEPTIDE RECEPTOR NUMBER ON RECEPTOR AFFINITY, COUPLING, DEGRADATION, AND MODULATION

The relationship between receptor number and agonist-induced intracell ular responses has been well studied in receptors coupled to adenylate cyclase; however, for receptors coupled to phospholipase C (PLC), ver y little is known about the effect of receptor number on receptor-medi ated processes. To explore this issue, we investigated the effect of t he number of receptors for gastrin-releasing peptide (GRP) on ligand a ffinity and on the ability to activate intracellular messengers [PLC, tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK))] an d cause receptor modulation (internalization, desensitization, down-re gulation) and ligand degradation. Three BALE 3T3 cell lines were made that stably expressed the gastrin-releasing peptide receptor (GRP-R) w ith receptor numbers varying by 280-fold (GRP-R-Low, GRP-R-Med, and GR P R-HI). Each cell line had the same affinity for agonist. The efficac y for bombesin to increase rt-l]inositol phosphates but not tyrosine p hosphorylation of p125(FAK) correlated well with receptor number. In c ontrast, the EC50 value for [H-3]inositol phosphate generation for bom besin was the same in each cell line. Receptor number did not alter in ternalization. In the absence of protease inhibitors, there was an inv erse correlation between receptor number and receptor down-regulation and desensitization. However, with protease inhibitors present, GRP-R- Med and GRP-R-Hi down-regulated significantly less than the GRP-R-Low. Similarly, GRP-R-Low desensitized significantly more than GRP-R-Med o r GRP-R-Hi. GRP-R-HI caused significantly greater ligand degradation t han GRP-R-Low, and protease inhibitors completely inhibited degradatio n by GRP-R-Low and inhibited degradation by 70% for GRP-R-Hi. In concl usion, we show that for the PLC-coupled GRP-R, receptor number had lit tle or no effect on binding affinity, potency for activating PLC, tyro sine phosphorylation of p125(FAK). Or extent of receptor internalizati on. In contrast, receptor number had an effect on ligand degradation, down regulation, desensitization, and efficacy of PLC activation witho ut altering the efficacy of tyrosine phosphorylation of p125(FAK) Thes e results demonstrate that the effect of receptor number differs for t he different functions mediated by the GRP receptor and differs from t hat reported for adenylate cyclase-coupled receptors such as receptors mediating the action of adrenergic agents, secretin, and opioids.