Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes.

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)