Gastrin and cholecystokinin (CCK) make up a family of important gastrointestinal and central nervous system peptide hormones and neurotransmitters. In the gastrointestinal system, these peptides become elevated in response to a meal and function to stimulate secretion and motility necessary to regulate the digestive process. CCK is highly expressed in the CNS where its function is less clear, although it has been implicated in satiety, anxiety and nociception. Historically, we have been interested in understanding the structural basis of the interaction of CCK and gastrin with their G protein- coupled receptors, CCKAR and CCKBR, respectively, and the regulation and signaling of these receptors. In the past year, we have focused in these areas and a novel area concerning gastrin regulation of renal fucntion: 1) CCKBRs expressed in neurons undergo rapid desensitization and we have shown in a number of transformed cell lines that CCKBRs undergo rapid internalization that is in large part dependent on serine and threonine residues in the cytoplasmic tail. To characterize the process of internalization and desensitization of CCKBRs, we studied the WT rat CCKBR stably expressed in CHO-K1 cells and an internalization impaired mutant CCKBR with all 10 potential Ser/Thr phosphorylation sites mutated to Ala, CCKBR(*S/T). Metabolic labeling with 32P orthophosphate confirmed that these mutated sites could account for all of ligand stimulated CCKBR phosphorylation. CCKBR(*S/T) internalization was markedly reduced to = 5% at 5 minutes compared to = 60% for WT CCKBR without any change in ligand affinity or signaling. To study short term desensitization and resensitization in the same cells in real time, CCK-8 stimulated changes in acidification rate (AR) were measured using a microphysiometer . No differences in desenstitization and resensitization were observed between CHO-K1 cells expressing WT versus CCKBR(*S/T) indicating that, unlike the CCKAR, the CCKBR undergoes rapid internalization and desensitization and that internalization is not important for short term desensitization or resensitization. 2) At present, there are no convenient sources of either pure primary cells or transformed cell lines expressing high levels of either CCKAR or CCKBR alone. In addition, receptors expressed in heterologous cells may not accurately reflect their function in native cells. Therefore, we developed a recombinant Semliki Forest Virus vector system for the delivery of native and mutant CCKRs as well as a variety of native and dominant negative signaling proteins for delivery into primary cultures of pancreatic acinar cells. In collaboration with Dr. John Polo at Chiron Corp., we have succeeded in producing large quantities of high titer virus using a Sindbis virus packaging cell line. In collaboration with John Rivera, NIAMS, we have developed a protocol to overcome the resistance to infection of primary cultures of pancreatic acini through the use of polyethylene glycol. We are now poised to begin a number of structure-function studies of CCKR signaling as well as dissect out the signaling pathways using a variety of dominant negative signaling proteins in native pancreatic cells. 3) It has been observed for more than a century that ingestion of a meal results in acute changes in renal function necessary to handle the absorbed nutrients. However, the mechanism for communication between the digestive and renal systems is unknown. To determine whether gastrin may serve as the mediator, receptors for gastrin (CCKB/gastrin receptor) were identified in proximal tubules of rat kidney. Gastrin, elevated by either a gavaged meal or direct renal infusion, stimulated an increase in urinary Na+ excretion and urine volume that is inhibited by the CCKBR/gastrin receptor-specific antagonist, L-365,260. Gastrin stimulated excretion of sodium and water and its reversal by L-365,260 were paralleled by CCKB/gastrin mediated inhibition and reversal of inhibition of renal tubular Na+-K+-ATPase activity, respectively. The identification and functional characterization of renal tubular gastrin receptors demonstrates the direct link between digestion and renal function mediated by the principle meal-stimulated gastrointestinal hormone, gastrin. - cholecystokinin, gastrin, CCK, receptors, phosphorylation, desensitization, kidney, SFV, virus,
