Negative regulation of parathyroid hormone (PTH)-activated phospholipase C by PTH/PTH-related peptide receptor phosphorylation and protein kinase A.
PTH binding to the PTH/PTHrP receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases receptor phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP receptor phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P<0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCbeta3 phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCbeta3 in which Ser1105 was mutated to alanine (PLCbeta3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP receptor phosphorylation and PKA. Furthermore, phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCbeta3 by PKA.
Tawfeek
Hesham A W
HA
Endocrine Unit, Massachusetts General Hospital, 55 Fruit Street, Thier 1051, Boston, Massachusetts 02114, USA. tawfeek@helix.mgh.harvard.edu
Abou-Samra
Abdul B
AB
D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D010738:Type C Phospholipases / Q000378:metabolism*
D007295:Inositol Phosphates; D050505:Mutant Proteins; D010281:Parathyroid Hormone; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1; C092449:inositol 1,2,3-trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11