Phospholipase D- and Protein Kinase C Isoenzyme-Dependent Signal Transduction Pathways Activated by the Calcitonin Receptor

Abstract

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product[ 3H]choline with an EC50 = 2.5 ± 0.3 × 10−8m, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 ± 1.0 × 10−8m). The hormone failed to induce release of [3H]phosphocholine and[ 3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-Cα, but not the δ isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-Cζ, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.