Stimulation by parathyroid hormone of a NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cbeta, and actin increases intracellular calcium in opossum kidney cells.
Parathyroid hormone (PTH) binds its cognate G-protein-coupled receptor (PTH1R) and signals through both adenylyl cyclase and phospholipase C (PLC). C-terminal determinants of the PTH1R interact with the Na+/H+ exchanger regulatory factor 1 (NHERF-1) by binding the first of two PDZ (psd95, discs-large, ZO-1) domains. Compared with wild-type opossum kidney (OK) cells, OKH cells, a sub-clone, do not display PTH-mediated increases of [Ca2+]i and express NHERF-1 at markedly lower levels. Stable expression of NHERF-1 in the OKH parent (OKH-N1) restores the PTH-mediated increase of [Ca2+]i that arises from an influx of extracellular calcium and is both PLC-dependent and pertussis toxin-sensitive. From a morphological perspective, NHERF-1 and the PTH1R co-localize to apical patches of OKH-N1 cells, an expression pattern that is absent in OKH cells and depends on a direct NHERF-1-PTH1R interaction in OKH-N1 cells. Actin and PLCbeta1 and -beta3 co-localize with NHERF-1 and the PTH1R in OKH-N1 cell apical patches. Actin is also an integral component of the NHERF-1-assembled complex because cytochalasin D disrupts apical localization of both NHERF-1 and the PTH1R and inhibits the PTH-mediated increase of [Ca2+]i. Expression of the first PDZ domain of NHERF-1 acts as a dominant-negative interactor by blocking apical localization of the PTH1R and inhibiting PTH-elicited increases of [Ca2+]i. Thus, NHERF-1 assembles a signaling complex in the apical domains of OK cells that contains the PTH1R, PLCbeta, and the actin cytoskeleton. Disruption of this complex blocks the PTH mediated increases of intracellular calcium.
Mahon
Matthew J
MJ
Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Segre
Gino V
GV
D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002460:Cell Line; D007527:Isoenzymes / Q000502:physiology*; D007668:Kidney / Q000502:physiology*; D009893:Opossums; D010281:Parathyroid Hormone / Q000502:physiology; D054799:Phospholipase C beta; D010750:Phosphoproteins / Q000502:physiology*; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers; D010738:Type C Phospholipases / Q000502:physiology*
D000199:Actins; D007527:Isoenzymes; D010281:Parathyroid Hormone; D010750:Phosphoproteins; D044168:Receptor, Parathyroid Hormone, Type 1; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D002118:Calcium