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 (Glu(19) --> Arg(19)) 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 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 alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(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 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 [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.
Takasu
H
H
Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA.
Gardella
T J
TJ
Luck
M D
MD
Potts
J T
JT
Bringhurst
F R
FR
D000262:Adenylyl Cyclases / Q000378:metabolism / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism* / Q000502:physiology
D008024:Ligands; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D018016:Receptors, Parathyroid Hormone; C400002:parathyroid hormone (1-28); D010738:Type C Phospholipases; D000262:Adenylyl Cyclases