PHOSPHOLIPASE-D-DEPENDENT AND PROTEIN-KINASE-C ISOENZYME-DEPENDENT SIGNAL-TRANSDUCTION PATHWAYS ACTIVATED BY THE CALCITONIN RECEPTOR

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor acti vating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimula ted dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [H-3]choline with an EC50 = 2.5 +/- 0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism ( EC50 = 4.5 +/- 1.0 x 10(-8) M). The hormone failed to induce release o f [H-3] phosphocholine and [H-3]glycerophosphocholine, ruling out acti vation of phosphatydilcholine-specific phospholipase C and phospholipa se A. Calcitonin stimulated phosphatidic acid, a product of phospholip ase D-dependent phosphatydilcholine hydrolysis. Activation of phosphol ipase D was confirmed by release of [H-3]phosphatydilethanol, a specif ic 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 phospholi pase C and of phospholipase D. Consequently, the protein kinase-C alph a, but not the delta isoenzyme, was cytosol-to-membrane translocated b y approximately 50% after 20 min exposure to calcitonin, whereas prote in kinase-C zeta, which was approximately 40% membrane-linked in unsti mulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying h igher 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 cassett e encoding for a 16-amino acid insert in the predicted first intracell ular loop. This modification is likely to prevent the calcitonin recep tor from associating to phospholipase-coupled signaling.