Na/H exchanger regulatory factors control parathyroid hormone receptor signaling by facilitating differential activation of G(alpha) protein subunits

Abstract

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [(35)S]GTPgammaS binding and G(alpha) subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of G(alpha)(q) but have no effect on stimulation of G(alpha)(i) or G(alpha)(s). In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both G(alpha)(q) and G(alpha)(i) but decrease stimulation of G(alpha)(s). Consistent with these functional data, NHERF2 formed cellular complexes with both G(alpha)(q) and G(alpha)(i), whereas NHERF1 was found to interact only with G(alpha)(q). These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.

FIGURE 1.

Specificity of PTH-induced [³⁵S]GTPγS binding to G protein subunit. A, cell membrane protein (20 μg) prepared from PS120-R or PS120-R-N2 cells was resolved on 10% SDS-polyacrylamide gels as described under “Experimental Procedures” for immunoblot analysis. Cell membrane PTHR expression was used as a loading control (55). B, membrane aliquots from PS120-R cells were incubated with [³⁵S]GTPγS and 100 nm PTH(1–34) in the presence or absence of 1 mm AMP-PCP or 100 μm unlabeled GTPγS for 5 min at 30 °C. Immunoprecipitation of [³⁵S]GTPγS bound to Gα_s subunit was measured as described under “Experimental Procedures.” Data are summarized as the mean ± S.E. of three independent experiments.

FIGURE 2.

Time- and concentration-dependent PTH induction of [³⁵S]GTPγS binding to Gα_s protein subunit. Cell membranes were prepared from PS120-R cells. Data are summarized as the mean ± S.E. of three independent experiments. A, time course of PTH-stimulated [³⁵S]GTPγs binding to Gα_s subunit was measured in the presence or absence of 100 nm PTH(1–34). B, concentration-dependent curve of PTH-stimulated [³⁵S]GTPγs binding to Gα_s subunit was measured in the presence of PTH for 5 min. Data are summarized as the mean ± S.E. of three independent experiments.

FIGURE 3.

NHERF2 inhibits PTH-stimulated Gα_s binding and increases both Gα_q and Gα_i binding in PS120-R cell membranes. A, equal amounts of membrane (Mem) and cytosolic (Cyt) proteins (20 μg) from PS120-R cells or PS120-R-N2 cells were immunoblotted with NHERF2 antibody. B, PTH-stimulated [³⁵S]GTPγs binding to Gα_s, Gα_q, or Gα_i protein was measured in PS120-R or PS120-R-N2 cell membranes. Data are summarized as the mean ± S.E. of four independent experiments. **, p < 0.01; ***, p < 0.001, compared with PS120-R plus PTH group. C, cell surface binding of ¹²⁵I-PTH(1–34) in PS120-R cells or PS120-R-N2 cells was measured as described under “Experimental Procedures.” Data are summarized as the mean ± S.E. of triplicate determinations.

FIGURE 4.

Effects of NHERF1 and NHERF2 on PTH-stimulated [³⁵S]GTPγS binding to Gα protein subunits in CHO-R cell membranes. A, equal amounts of membrane proteins (20 μg) from CHO-R cells transiently transfected with vector, His-NHERF1, or His-NHERF2 were immunoblotted (IB) with His antibody. Cell membrane PTHR expression was used as a loading control. B, PTH-stimulated [³⁵S]GTPγs binding to Gα_s, Gα_q, or Gα_i protein was measured in the cell membranes of CHO-R cells transiently transfected with vector, His-NHERF1, or His-NHERF2. Data are summarized as the mean ± S.E. of four independent experiments. **, p < 0.01; ***, p < 0.001 compared with vector plus PTH group. C, cell surface binding of ¹²⁵I-PTH(1–34) in CHO-R cells transiently transfected with vector, His-NHERF1, or His-NHERF2 was measured. Data are summarized as the mean ± S.E. of triplicate determinations.

FIGURE 5.

Effects of NHERF1 and NHERF2 on PTH-stimulated [³⁵S]GTPγS binding to Gα protein subunits in HEK-293R cell membranes. A, GST-tagged C-terminal 22 amino acids of PTH1R (PTHR-ctETVM) and its mutant of PTHR-ctETVA were overlaid with His-tagged PDZ domain of NHERF1 or NHERF2 on nylon membranes as described under “Experimental Procedures.” A representative overlay assay shows that PTH1R interacts with both PDZ domains of NHERF1 or NHERF2. B, equal amounts of membrane proteins (20 μg) from HEK-293R cells transiently transfected with HA-PTHR-ETVM or HA-PTHR-ETVA were immunoblotted (IB) with HA antibody. C, PTH-stimulated [³⁵S]GTPγs binding to Gα_s, Gα_q, or Gα_i protein was measured in the cell membranes of HEK-293R cells transiently transfected with HA-PTHR-ETVM or HA-PTHR-ETVA. Data are summarized as the mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01 compared with PTHR-ETVM group. D, equal amounts of membrane proteins (20 μg) from HEK-293R cells transiently transfected with scrambled shRNA, NHERF1 shRNA (shNHERF1), or NHERF2 shRNA (shNHERF2) were immunoblotted with either NHERF1 or NHERF2 antibody. E, PTH-stimulated [³⁵S]GTPγs binding to Gα_s, Gα_q, or Gα_i protein was measured in the cell membranes of HEK-293R cells transiently transfected with scrambled shRNA, shNHERF1 or shNHERF2. Data are summarized as the mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.01, compared with scrambled plus PTH group. F, rescue experiments were performed by cotransfection with shNHERF1, shNHERF2, and plasmid constructs of WT-NHERF1, WT-NHERF2, or mutated NHERF1 and NHERF2 (resNHERF1 and resNHERF2) that are refractory to cleavage by their respective shRNA as described under “Experimental Procedures.” PTH-stimulated [³⁵S]GTPγs binding to Gα_s, Gα_q, or Gα_i protein was measured. Data are summarized as the mean ± S.E. of four independent experiments. **, p < 0.01, versus respective scrambled plus vector group. G, Gα_i activation measured by real time FRET. FRET was performed on HEK-293 cells cotransfected with shNHERF1 or shNHERF2 and plasmid constructs of PTHR-ETVM, PTHR-ETVA, ^YFPGα_i and bimolecular fluorescence complementation β₁γ₂ complexes as described under “Experimental Procedures.” Data are summarized as the normalized FRET mean ± S.E. of three independent experiments.

FIGURE 6.

NHERF2 but not NHERF1 binds Gα_i. A, PTH increased the interaction of Gα_q protein with NHERF1 or NHERF2. CHO-R cells were transfected with His-NHERF1 or His-NHERF2. His-tagged proteins were precipitated with Ni-NTA-agarose. The precipitated protein was then immunoblotted (IB) with Gα_q antibody. B, PTH increased the interaction of Gα_i proteins with NHERF2. CHO-R cells were transfected with His-NHERF1 or His-NHERF2. His-tagged proteins were precipitated with Ni-NTA-agarose or endogenous Gα_i protein was precipitated with Gα_i antibody. The precipitated proteins were immunoblotted with either Gα_i or His antibody. C, interaction of Gα_q or Gα_i subunits with NHERF1 or NHERF2 in CHO cells was measured and described as above. Data are representative of three independent experiments. IP, immunoprecipitated.

FIGURE 7.

Effects of NHERF1 and NHERF2 on PTH-stimulated adenylyl cyclase activity and [Ca²⁺]_i. A, effects of NHERF1 and NHERF2 on PTH-stimulated adenylyl cyclase activity. HEK-293R cells were transfected with scrambled shRNA, shNHERF1, or shNHERF2. Pertussis toxin (PTX) (100 ng/ml) was added for 16 h as indicated. Cells were treated with 100 nm PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean ± S.E. of four independent experiments. **, p < 0.01 compared with scrambled shRNA plus PTH group. B, effects of NHERF1 and NHERF2 on PTH-induced [Ca²⁺]_i. HEK-293R cells were treated as the same as A. PTH (100 nm)-stimulated [Ca²⁺]_i was measured as described under “Experimental Procedures.” Data are summarized as the mean ± S.E. of three independent experiments. The effects of scrambled shRNA, shNHERF1, and shNHERF2 on [Ca²⁺]_i in the presence or absence of pertussis toxin were shown in top, middle, or bottom panel, respectively.