Amino-Terminal Modifications of Human Parathyroid Hormone (PTH) Selectively Alter Phospholipase C Signaling via the Type 1 PTH Receptor:  Implications for Design of Signal-Specific PTH Ligands

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1−34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29−32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1−34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1−34) but were coordinately restored when a binding-enhancing substitution (Glu19 → Arg19) was placed within hPTH-(1−28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly1 for Ser1 in hPTH-(1−34) and was eliminated entirely by removing either residue 1 or the α-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly1,Arg19]hPTH-(1−28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28−31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1−34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly1,Arg19]hPTH-(1−28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.