A midregion fragment of PTH-related protein (PTHrP), which is intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2+](i)) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (b) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 μM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67- 86)NH2 on [Ca2+](i) were additive. The inhibitory PTH analog, [D- Trp12,Tyr34]bovine, PTH-(7-34)NH2, attenuated the [Ca2+](i) response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2, did not bind to SqCC/Y1 cells, and PTHrP- (67-86)NH2 did not compete for binding of 125I-labeled [Tyr36]PTHrP- (1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP-(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 ± 0.1-fold over the control value, with an EC50 of 1.5 ± 1.2 nM. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 μM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation el cAMP formation by isoproterenol (1 μM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation era receptor protein that mediated a [Ca2+](i) response to PTHrP-(67-86)NH2, we performed expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 μM PTHrP-(67-86)NH2. Clear increases in [Ca2+](i) were detected in mRNA- injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(6786)NH2 treatment on fibronectin secretion from SqCC/Y1 cells. A significant 3.5-fold increase in fibronectin secretion into conditioned medium was observed when SqCC/Y1 cells were exposed to 100 nM PTHrP-(67- 86)NH2, and this effect was dose dependent, with an EC50 of 0.1 nM. We conclude that PTHrP-(67-86)NH2 activates phospholipase C-dependent pathways in SqCC/Y1 cells through a receptor distinct from that activated by PTHrP- (1-36) in the same cells. As a midregion secretory fragment of PTHrP has been partially purified from several different cell types, this receptor may have broad biological significance.
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