Cleavage of N-terminus of polycystin-1 increases calcium permeability of polycystin-1/2 receptor channel complexes
Runping Wang, Danish Idrees, Mohammad Amir, Biswajit Padhy, Jian Xie, Chou-Long Huang
Runping Wang, Danish Idrees, Mohammad Amir, Biswajit Padhy, Jian Xie, Chou-Long Huang
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Research Article Cell biology Nephrology

Cleavage of N-terminus of polycystin-1 increases calcium permeability of polycystin-1/2 receptor channel complexes

Abstract

Mutations on genes encoding polycystin-1 (PC1) and PC2 cause autosomal-dominant polycystic kidney disease. How these 2 proteins work together to exert anticystogenesis remains elusive. PC1 resembles adhesion G-protein coupled receptors and undergoes autocleavage in the extracellular N-terminus to expose a hidden “stalk” region, which is hypothesized to act as a “tethered agonist.” Here, we show that WT PC1 and PC2 formed functional heteromeric channel complexes in Xenopus oocytes with different biophysical properties from PC2 homomeric channels. Deletion of PC1 N-terminus, which exposed the stalk, increased calcium permeability in PC1/PC2 heteromers that required the presence of stalk. Extracellular application of synthetic stalk peptide increased calcium permeation in stalkless PC1/PC2. Application of Wnt9B protein increased calcium permeability in PC1/PC2 but not in heteromers containing cleavage-resistant mutant PC1. Wnt9B interacted with N-terminal leucine-rich repeat (LRR) of PC1. Pretreatment with LRR blunted the increase in calcium permeability by Wnt9B. Thus, PC1 and PC2 form receptor-channel complexes that is activated by exposure of the stalk region following ligand binding to the PC1 N-terminus. The stalk peptide acts as a tethered agonist to activate PC1/PC2 by affecting ion selectivity of the complexes.

Authors

Runping Wang, Danish Idrees, Mohammad Amir, Biswajit Padhy, Jian Xie, Chou-Long Huang

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Figure 2

PC1 increases surface expression and K+ permeability of PC2.

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PC1 increases surface expression and K+ permeability of PC2. (A) I-V rel...
(A) I-V relationship of currents from oocytes expressing PC2 alone or sPC1/PC2 recorded in100 mM NaCl. (B) Mean ± SEM for inward and outward currents at –100 and 100 mV in oocytes injected with vehicle or mRNA for PC2, sPC1 or both; n = 10, 5, 8, and 8, respectively. (C) Oocytes injected with vehicle or mRNA for a HA-tagged PC2 (PC2302HA), with or without sPC1. PC2302HA contains an extracellular HA tag engineered to replace amino acids 302–306 of the TOP domain (second extracellular loop) of PC2. Top: representative Western blot of total oocyte lysates probed with anti-PC2 and anti–β actin antibody. Bottom: representative immunofluorescence (IF) staining images using anti-HA antibody extracellularly in nonpermeabilized oocytes. IF staining in oocytes injected with sPC1 alone was not different from vehicle-injected (not shown). (D) Mean ± SEM of normalized IF intensity of surface expression of PC2302HA in control (vehicle), PC2302HA, and sPC1/ PC2302HA-injected oocytes as in C (n = 9, 11, and 11, respectively). IF intensity was normalized to PC2302HA-injected oocytes. (E–H) Oocytes expressing PC2 or sPC1/PC2 were recorded in 100 mM NaCl, KCl, CaCl2, or NMDGCl. (E) Representative I-V with enlarged curves showing reversal potentials. (F) Mean ± SEM for inward and outward currents at –100 and 100 mV, n = 7 and 8 for PC2 and sPC1/PC2. (G) Reversal potentials. (H) Calculated relative permeability PX/PY. *P < 0.01 for PC2 versus sPC1/PC2. Two-tailed unpaired Student’s t test for B and D–F. In all panels, experimental number (n) is number of oocytes as shown by scatter plots. All experiments were repeated 2 or more times with similar results.