Volume 66, Pages S13-S19 (October 2004)

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Volume 66, Pages S13-S19 (October 2004) PTH revisited12  Peter A. Friedman  Kidney International  Volume 66, Pages S13-S19 (October 2004) DOI: 10.1111/j.1523-1755.2004.09103.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Parathyroid hormone (PTH) effects on [Ca2+]i in single proximal or distal tubule cells. At 2 minutes, 10 nmol/L PTH1-34 was added to the bath. Modified from55. Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Effects of PTH peptides on PTH1R internalization in proximal (PCT) and distal (DCT) kidney tubule cells. (A) PTH(1–34) internalizes the PTH1R in PCT and DCT cells, albeit with somewhat different kinetics. (B) PTH(7–34) avidly promotes PTH1R endocytosis in DCT cells, but has no effect on PCT cells. (C) Full-length PTH circulating peptides PTH(1–84) and PTH(7–84) exert effects comparable to their shorter synthetic analogues (i.e., both induce receptor sequestration in DCT cells). Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Na/H exchanger regulatory factor (NHERF) expression inhibits PTH/PTHrP (PTH1R) endocytosis in distal tubule cells. The results show the effect of 10-7 mol/L PTH(1–34) or PTH(7–34) on PTH1R internalization after 15 minutes in the absence or presence of NHERF1. **P < 0.01 vs. distal kidney tubule cells (DCT). Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Dominant-negative Na/H exchanger regulatory factor (dnNHERF) permits PTH(7–34) to internalize the PTH/PTHrP (PTH1R) in proximal tubule cells. The effect of 10-7 mol/L PTH(7–34) on PTH1R internalization is shown after 15-minute stimulation in cells transiently transfected with dnNHERF1 or empty vector. Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Coimmunoprecipitation of Na/H exchanger regulatory factor (NHERF1) with PTH1R(ETVM)-EGFP and PTH1R(ETVA)-EGFP. Human embryonic kidney (HEK)-293 cells transfected with the indicated constructs. Immunoprecipitation experiments were performed by incubating the cell lysates with an EGFP-specific polyclonal antibody. Immunoblotting was performed with an HA-specific monoclonal antibody (upper panel). The amount of immunoprecipitated EGFP-tagged receptor in each sample was verified by immunoblotting with the EGFP-specific polyclonal antibody (middle panel). The quantity of HA-NHERF1 present in each cell extract was evaluated by immunoblotting with the HA-specific monoclonal antibody (lower panel). Modified from48. See text for abbreviations. Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Na/H exchanger regulatory factor (NHERF1) effects on parathyroid hormone (PTH)-induced internalization in distal tubule cells expressing either the wild-type PTH/PTHrP (PTH1R) PDZ-recognition motif or the mutated ETVA PDZ-binding domain. The results depict the effect of 10-7 mol/L PTH(1–34) or PTH(7–34) for 15 minutes. **P < 0.01 vs. wild-type PTH1R. See text for abbreviations. Kidney International 2004 66, S13-S19DOI: (10.1111/j.1523-1755.2004.09103.x) Copyright © 2004 International Society of Nephrology Terms and Conditions