The protein kinase-C activation domain of the parathyroid hormone.

Abstract

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.

Dual Signaling and Ligand Selectivity of the Human PTH/PTHrP Receptor

“…As expected, stimulation of PLC by hPTHrP(1-36) was equivalent to that of hPTH(1-34) in both HKRKB28 (at 4 minutes) and HKRKB7 (at 30 minutes). Activation of PKC by hPTH at very low concentrations has been described, (17) but we observed no stimulation of PLC by this peptide at concentrations between 1 pM and 10 nM (data not shown).…”

“…Much effort has focused upon attempts to modulate PTHR signaling through use of PTH analogs, such as PTH or PTH , which may selectively activate one or the other of the two major PTHR signaling effectors, AC and PLC (10)(11)(12)(13)(14)(15)(16) For example, selective activation of the AC/protein kinase A (PKA) pathway by intermittently administered PTH(1-31), a putative ACselective PTHR agonist, was reported to account entirely for the anabolic response of bone in vivo, whereas hPTH(3-34), which does not activate AC but does stimulate protein kinase C (PKC) in bone cells, was inactive in vivo. (14,(16)(17)(18) Others have employed PTH to probe the involve-ment of PKC in the regulation of renal ion transporters and enzymes by PTH in rat and opposum kidney cells. (10,12,15) The ligand selectivity and signaling properties of the human PTHR (hPTHR) have not been widely studied.…”

“…(21) These and other studies (24) have emphasized the possible involvement of PLC-independent mechanisms in generating some intracellular calcium responses via the hPTHR. Reports of the actions of PTH , a putative PLC/PKC-selective analog, (10,11,17,18,25) via recombinant hPTHRs in these systems, are contradictory. Direct measurements of PTH(3-34)-stimulated PLC activation via expressed hPTHRs have not been reported.…”

Structure-Activity Relation of NH2-terminal Human Parathyroid Hormone Fragments

“…A single receptor was shown to stimulate intracellular accumulation of both cAMP and inositol triphosphates (39). PTH has the concentration-dependent ability to stimulate two separate signal pathways (9), and different sequential regions of the hormone may be responsible for initiation of the adenylate cyclase and the phospholipase C activating pathway. The existence of these multiple pathways is possibly reflected by the fact that hPTH-(2-37) is virtually inactive in the adenylate cyclase assay but can induce substantial hypercalcemia in the in vivo model (Fig.…”

“…4 and references therein). This indicates that hPTH utilizes other second messengers in addition to cAMP for signal transduction and possibly additional receptors in vivo (9). Furthermore, hPTH is stimulating cell proliferation in skeletal derived cell cultures (10,11) as well as DNA synthesis in chondrocytes (12).…”