Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: Implicationsfor 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 p hospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essent ial for AC activation. Ligand domains required for activation of PLC, PKC, and other effecters have been less well-defined, although some studies in r odent 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 cel ls that stably express abundant transfected human or rat PTH/PTHrP receptor s. Phospholipase C signaling and ligand-binding affinity were reduced by ca rboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restore d when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed wi thin hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituti ng Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alt er binding affinity. These findings led to design of an analogue, [Gly(1),A rg(19)]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 hP TH-(28-31), contributes to PLC activation through effects upon receptor bin ding but is not required for full PLC activation. The N-terminal determinan ts of AC and PLC activation in hPTH-(1-34) overlap but are not identical, a s subtle modifications in this region may dissociate activation of these tw o effecters. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, shoul d prove useful in dissociating AC- from PLC-dependent actions of PTH.