Chronic desensitization and down-regulation of the gastrin-releasing peptide receptor are mediated by a protein kinase C-dependent mechanism.
The cellular basis of down-regulation and desensitization in phospholipase C-linked receptors is unclear. Recent studies with some receptors suggest that elements in the carboxyl terminus of the receptor are important in mediating these processes. Three mutant gastrin-releasing peptide receptors (GRP-R) were studied: one whose last 37 carboxyl-terminal amino acids were eliminated (construct MGT346); one that replaced all of the carboxyl-terminal Ser and Thr eliminated in MGT346 with Ala, Asn, or Gly (construct JF1); and one that selectively replaced the Ser and Thr of the protein kinase C consensus sequence (PKC-CS) located within the same region with alanine (construct TS360AA). Desensitization was assessed by measuring the ability to activate phospholipase C and increase cellular [3H]inositol phosphates, or increase [Ca2+]i, after pre-exposure to 3 nM bombesin for 24 h. Wild-type GRP-R was maximally desensitized and down-regulated after a 24-h exposure to 3 nM bombesin, and removal of the PKC-CS alone markedly attenuated each process. Elimination of additional serines and threonines by truncation (MGT346) or replacement (JF1) did not decrease down-regulation or desensitization further. To confirm the necessity of second messenger activation in mediating down-regulation, we further investigated two additional mutant GRP-R that bound agonist with high affinity but fail to activate phospholipase C (constructs R139G and A263E). Neither construct underwent significant down-regulation. Removal of all GRP-R carboxyl-terminal Ser or Thr, either by MGT346 or JF1, reduced internalization by > 80%, whereas elimination of the PKC-CS in TS360AA only attenuated internalization by 21 +/- 2%. These data suggest that activation of the distal carboxyl-terminal PKC-CS is essential for chronic desensitization and down-regulation of the GRP-R, and provide no evidence for involvement of second messenger-independent processes. In contrast, internalization is equally regulated by both second messenger-dependent and independent processes.
Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804.
R V
RV
Benya
T
T
Kusui
J F
JF
Battey
R T
RT
Jensen
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001839:Bombesin / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D016384:Consensus Sequence; D015536:Down-Regulation; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011493:Protein Kinase C / Q000378:metabolism*; D018004:Receptors, Bombesin / Q000096:biosynthesis / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection
D007295:Inositol Phosphates; D018004:Receptors, Bombesin; D011493:Protein Kinase C; D001839:Bombesin