F. Naro et al., PHOSPHOLIPASE-D-DEPENDENT AND PROTEIN-KINASE-C ISOENZYME-DEPENDENT SIGNAL-TRANSDUCTION PATHWAYS ACTIVATED BY THE CALCITONIN RECEPTOR, Endocrinology, 139(7), 1998, pp. 3241-3248
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.