Mapping of a carboxyl terminal active site of parathyroid hormone by calcium imaging

We recently showed that the C-terminal fragment PTH (52-84) effectively increases intracellular free calcium ([Ca2+](i)) in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1-34). Here we characterize the active site on C-terminal PTH (52-84) with respect to calcium (Ca2+)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73-76 to be the core region for increasing [Ca2+](i). Ryanodine (1 mu M), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2-5 mu M), agents that interfere with intracellular Ca2+ release, all failed to block PTH (52-84) induced [Ca2+](i) increases. Depletion of extracellular calcium ([Ca2+](o)) blocked PTH (52-84) induced [Ca2+](i) increases, indicating a transmembrane Ca2+ influx. In contrast to voltage-gated and Ca2+ release activated Ca2+ influx, PTH (52-84) evoked Ca2+ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73-76 are essential for activation of a nickel-insensitive Ca2+ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.