PTH activates multiple second messengers in its target cells, but the level at which the hormonal signal splits into different pathways is still unknown. To achieve insights on this issue, we have studied the structure-function relationship of PTH by analyzing the effects of bovine PTH-(1-34) [
bPTH-(1-34)] and PTH fragments truncated at the N-terminus on the intracellular
calcium concentration [( Ca2+]i) and cAMP production in the rat
osteogenic sarcoma cell line UMR 106-01. [Ca2+]i was measured in single cells using
fura-2. When exposed to 10(-7) M bPTH-(1-34), 20% of the cells responded with a transient increase in [Ca2+]i of variable amplitude. Equimolar doses of bPTH-(2-34), propionyl bPTH-(2-34) [(pbPTH-(2-34)], and
bPTH-(3-34) also transiently increased [Ca2+]i, whereas both [tyrosine34]
bPTH-(7-34)
amide [
bPTH-(7-34)] and bPTH-(30-34) were ineffective. The amplitude of the [Ca2+] i transients was dose-dependent, with threshold concentrations of 10(-10) M for
bPTH-(1-34) and bPTH-(2-34), and 10(-9) M for
bPTH-(3-34). The response rate to the active
peptides ranged between 10-30%, without a clear dose-relatedness. A second addition of 10(-7) M
bPTH-(1-34) to cells prestimulated with equimolar doses of bPTH-(2-34), pbPTH-(2-34), or
bPTH-(3-34) produced another transient, whereas after exposure to 10(-7) M
bPTH-(1-34), the cells were completely desensitized to a second homologous stimulation, suggesting that the binding affinity of the truncated
peptides for the PTH receptor is lower than that of the intact
bPTH-(1-34) fragment. In addition, both
bPTH-(1-34) and bPTH-(2-34) dose-dependently stimulated cAMP production, but the former was more potent (ED50 = 10(-9) vs. 10(-7) M, respectively). On the contrary, pbPTH-(2-34),
bPTH-(3-34), and
bPTH-(7-34) had no effect on cAMP. Pretreating the cells with
pertussis toxin to enhance cAMP responses via inhibition of Gi potentiated the effect of
bPTH-(1-34) and bPTH-(2-34) and disclosed weak but detectable agonist action of pbPTH-(2-34). These results indicate that specific domains of the PTH molecule are linked to activation of different second messenger pathways; while the first two
amino acids are indispensable for activating the cAMP system, generation of the [Ca2+]i signal appears to involve a longer domain, including the
amino acid residue in position 3.