Colin A. Syme

Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50)

Parathyroid hormone (PTH) regulates extracellular calcium homeostasis through the type 1 PTH receptor (PTH1R) expressed in kidney and bone. The PTH1R undergoes β-arrestin/dynamin-mediated endocytosis in response to the biologically active forms of PTH, PTH-(1–34), and PTH-(1–84). We now show that amino-truncated forms of PTH that do not activate the PTH1R nonetheless induce PTH1R internalization in a cell-specific pattern. Activation-independent PTH1R endocytosis proceeds through a distinct arrestin-independent mechanism that is operative in cells lacking the adaptor protein Na/H exchange regulatory factor 1 (NHERF1) (ezrin-binding protein 50). Using a combination of radioligand binding experiments and quantitative, live cell confocal microscopy of fluorescently tagged PTH1Rs, we show that in kidney distal tubule cells and rat osteosarcoma cells, which lack NHERF1, the synthetic antagonist PTH-(7–34) and naturally circulating PTH-(7–84) induce internalization of PTH1R in a β-arrestin-independent but dynamin-dependent manner. Expression of NHERF1 in these cells inhibited antagonist-induced endocytosis. Conversely, expression of dominant-negative forms of NHERF1 conferred internalization sensitivity to PTH-(7–34) in cells expressing NHERF1. Mutation of the PTH1R PDZ-binding motif abrogated interaction of the receptor with NHERF1. These mutated receptors were fully functional but were now internalized in response to PTH-(7–34) even in NHERF1-expressing cells. Removing the NHERF1 ERM domain or inhibiting actin polymerization allowed otherwise inactive ligands to internalize the PTH1R. These results demonstrate that NHERF1 acts as a molecular switch that legislates the conditional efficacy of PTH fragments. Distinct endocytic pathways are determined by NHERF1 that are operative for the PTH1R in kidney and bone cells.

An NH2-terminal multi-basic RKR motif is required for the ATP-dependent regulation of hIK1.

We previously demonstrated that the ATP/PKA‑dependent activation of the human intermediate conductance, Ca2+‑activated K+ channel, hIK1, is dependent upon a C‑terminal motif. The NH2‑terminus of hIK1 contains a multi‑basic 13RRRKR17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation (15RKR17/AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole‑cell patch‑clamp. However, cell‑surface -immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the 15RKR17/AAA mutation we performed inside‑out patch clamp recordings where we observed no difference in Ca2+ affinity between the wild‑type and mutated channels. However, in contrast to wild‑type hIK1, channels expressing the 15RKR17/AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15RKR17/AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15RKR17/AAA mutation resulted in a four‑fold lower channel open probability (Po), in the presence of saturating Ca2+ and ATP, compared to wild‑type hIK1. In conclusion, these results represent the first demonstration for a role of the NH2‑terminus in the second messenger‑dependent regulation of hIK1 and, in -combination with our previous findings, suggest that this regulation is dependent upon a close NH2/C‑terminal association.