Characterization of interactions of Na+/H+ exchanger regulatory factor-1 with the parathyroid hormone receptor and phospholipase C.
The Na(+)/H(+) exchanger regulatory factor-1 (NHERF1) is a molecular scaffold important for the signaling of the G-protein coupled receptor for the parathyroid hormone (PTH1R). The two PDZ (PSD-95, Discs-large, ZO1) domains of NHERF1 through association with the C-termini of PTH1R and phospholipase C enhance the signaling pathway associated with PTH. To examine these interactions, we have produced the individual PDZ1 and PDZ2 domains as well as the tandem PDZ1-PDZ2 domains (PDZ12) of NHERF1 and have characterized the binding affinities of the C-terminal motifs of PTH1R and PLCbeta using fluorescence anisotropy. Circular dichroism indicates that the PDZ1 and PDZ2 are properly folded. Based on fluorescence anisotropy we find that the C-terminus of PTH1R, containing ETVM, has similar affinities (approximately 10 microm) for both PDZ1 and PDZ2. The PTH1R displayed reduced binding affinity for the tandem PDZ12 (16 microm) compared with the individual domains or a solution of equal molar concentrations of PDZ1 and PDZ2 (5.8 microm), suggesting negative cooperativity between the PDZ domains or intervening region. The C-termini of PLCbeta (both beta1 and beta2 isozymes were examined, containing DTPL and ESRL, respectively) displayed a diminished affinity for PDZ2 (approximately 30 microm) over that of PDZ1 (approximately 8 microm). Finally, we demonstrate trans PDZ1-PDZ2 association that is enhanced in the presence of the C-terminus of PTH1R or PLCbeta, suggesting oligomerization of NHERF as a mode for enhancing the signaling associated with PTH.
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Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.
Mierke
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D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005454:Fluorescence Polarization; D051379:Mice; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D010738:Type C Phospholipases / Q000378:metabolism*
D010750:Phosphoproteins; D018016:Receptors, Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D010738:Type C Phospholipases