Zheng W et al. (2015), A novel PKD2L1 C-terminal domain critical for t...

XB-ART-50284

Sci Rep 2015 Jan 12;5:9460. doi: 10.1038/srep09460.

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A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

Zheng W , Hussein S , Yang J , Huang J , Zhang F , Hernandez-Anzaldo S , Fernandez-Patron C , Zeng H , Tang J , Chen XZ .

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As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

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Species referenced: Xenopus
Genes referenced: ctrl dtl pkd1 pkd1l1 pkd1l3 pkd2l1 pycard shh

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	Figure 1. Oligomers of PKD2L1 in mouse tissues and human cell lines.(A) Detection of PKD2L1 in mouse kidney, testis, and brain tissues by WB. Samples were prepared with SDS loading buffer under the non-reducing and reducing (supplemented with 5% 2-ME or 0.5â€…M DTT) conditions. PKD2L1 was detected with an antibody from Abnova. Na/K ATPase was used as a loading control. Detection of over-expressed human PKD2L1 in HEK (B) and HeLa cells (C). pcDNA3.1(+) containing human PKD2L1 or empty vector (Ctrl) was transfected into HEK and HeLa cells. Cell lysates were collected 48â€…hr after transfection. Samples for SDS-PAGE were prepared under non-reducing or reducing condition. (D) Detection of over-expressed human PKD2L1 in HEK cells under non-reducing and reducing conditions. Samples were prepared with or without treatment by 10â€…mM NEM. (E) WB analysis after BN-PAGE of Flag-tagged PKD2L1 over-expressed in HeLa cells. Samples were prepared with or without 2.5% SDS, or with 2.5% SDS plus 0.1â€…M DTT. An anti-Flag antibody was used for WB detection. Putative PKD2L1 monomer, dimer and trimer are indicated.
	Figure 2. Roles of the human PKD2L1 N- and C-termini in its channel activity and oligomerization.(A) Representative whole-cell current tracings obtained from Xenopus oocytes expressing PKD2L1 WT, mutant Î´CT (deletion of E566-S805) or Î´NT (deletion of M1-Y96) using the two-microelectrode voltage clamp technique. Oocytes were voltage clamped at âˆ’50â€…mV. Data from a water-injected oocyte served as a negative control (Ctrl). Currents were measured using standard extracellular solution (pH 7.5) (7.5) or standard extracellular solution containing 5â€…mM CaCl2 (7.5+Ca). (B) Averaged currents obtained from oocytes expressing PKD2L1 WT, Î´CT, Î´NT or water (Ctrl). Currents were averaged from different numbers of oocytes, as indicated. â€˜***' indicates p â‰¤ 0.001 when compared with the WT data. (C) Representative currentâ€“voltage relationship curves obtained using a voltage ramp protocol, as indicated, before (7.5) and after (7.5+Ca) addition of 5â€…mM CaCl2. (D) Representative immunofluorescence data showing the plasma membrane expression of PKD2L1 WT, Î´CT and Î´NT in oocytes. (E) Averaged and normalized surface expression of PKD2L1 WT, Î´CT and Î´NT in oocytes. Surface expressions were averaged from indicated numbers of oocytes and normalized to that of PKD2L1 WT. (F) WB detection of Flag-tagged human PKD2L1 WT, Î´NT and Î´CT over-expressed in HeLa cells under the non-reducing condition. A band (indicated by an arrow) that is unlikely a dimer based on its size remained unaccounted for was detected with the CT deletion.
	Figure 3. Identification of domain C1 of PKD2L1 critical for its trimerization.(A) WB detection of over-expressed Flag-tagged PKD2L1 WT, without or with 10â€…mM NEM treatment, or mutants 4Ã—C-A (quadruple C38A, C60A, C70A and C74A mutations), C38A and 3Ã—C-A (triple C60A, C70A and C74A mutations) under non-reducing condition. (B) WB analysis after BN-PAGE of Flag-tagged PKD2L1 WT and Î´NT over-expressed in HeLa cells. WT samples were prepared with or without 2.5% SDS, or with 2.5% SDS plus 10â€…mM NEM. Putative PKD2L1 monomer, dimer and trimer are indicated. (C) Schematic illustration of PKD2L1 membrane topology. TMs, pore-loop, C1, CC2, EF-hand, coiled-coil domains, and positions of residues Y96, E566 and T622 are indicated. (D) WB detection of Flag-tagged PKD2L1 mutants Î´NT/Î´CT (M1-Y96 and E566-S805 double deletion) and Î´NT/T622X (M1-Y96 and T622-S805 double deletion) over-expressed in HeLa cells under the non-reducing condition. Two unknown bands similar to one in Fig. 2F remained unaccounted for. (E) WB detection of over-expressed Flag-tagged PKD2L1 mutants in HEK or HeLa cells. All constructs were made from Î´NT. These included Ctrl (Î´NT), Î´C1 (C1, K575-T622, deletion from Î´NT), Î´CC2 (CC2, G699-W743, deletion from Î´NT), or Î´C1/Î´CC2 (C1 and CC2 double deletion from Î´N). (F) Amino acid sequence alignment of PKD2L1 C1 from indicated species. National Center for Biotechnology Information accession number for sequences used here are NP_057196 (human), NP_852087 (mouse), XP_426509 (chicken), XP_005637930 (dog), XP_002698535 (cattle), and XP_001168415 (chimpanzee). Identical residues among the species are indicated by black bold letters.
	Figure 4. Identification of domain C1 of PKD2L1 critical for its channel function.(A) Representative whole-cell current tracings obtained from Xenopus oocytes expressing PKD2L1 truncation mutant T622X, S581X, or deletion mutant Î´C1 (C1 deletion from PKD2L1 WT) using the two-microelectrode voltage clamp technique under similar experimental conditions as those for Fig. 2. (B) Averaged currents obtained from oocytes expressing PKD2L1 WT or truncation/deletion mutants, as indicated. Currents at âˆ’50â€…mV were averaged from indicated numbers of oocytes and normalized to that of PKD2L1 WT. Water-injected oocytes were used as control (Ctrl). â€˜***' indicates P â‰¤ 0.001 when comparing with â€œWTâ€. (C) Representative currentâ€“voltage relationship curves obtained using a voltage ramp protocol, as indicated in Fig. 2C, before (7.5) and after (7.5+Ca) addition of 5â€…mM CaCl2. (D) Representative immunofluorescence data showing the plasma membrane expression of mutants T622X, S581X and Î´C1 expressed in oocytes, or those injected with water (Ctrl). (E) Surface membrane expression of mutants T622X, S581X and Î´C1 were averaged from the indicated numbers of oocytes and normalized to that of PKD2L1 WT.
	Figure 5. Further evidences for C1 involvement in trimerization.(A) Effect of C1 on the interaction between two PKD2L1 proteins assessed by co-IP. Left panel: representative data showing the interaction of GFP-PKD2L1 (GFP-WT) with Flag-T622X or Flag-K575X in HeLa cells. GFP-WT was first transfected, followed by transfection of T622X and K575X 12âhr later. Right panel: representative input data by WB showing the expression of WT, T622X and K575X. (B) Data from experiments in panel A were quantified, averaged, normalized, and then compared by paired t-test (*P â¤ 0.001, N = 3). (C) Coomassie blue staining of purified PKD2L1 C-terminus (CT, E566-S805), CT-ÎC1 (C1 deletion from CT), and CT-Îdouble (C1 and CC2 double deletion from CT) from E. coli. Data using crude lysates are shown on their left (lanes 1, 2 and 3). (D) Analysis of size distribution of purified CT, CT-ÎC1, and CT-Îdouble fragments by dynamic light scattering experiments. (E) Coomassie blue staining analysis of BN-PAGE or SDS-PAGE of purified protein GFP-C1 or GFP-Ctrl (human 4EBP1 M1-T50 fragment). Putative GFP-C1 trimer is indicated by an arrow on BN-PAGE. The faint bands below and above the putative trimer are indicated with stars. (F) Left panel: WB detection of over-expressed Flag-ÎNT with co-expression of HA-tagged blocking peptide C1 (HA-C1) or control peptide T622-E675 (HA-Ctrl) in HeLa cells under the non-reducing condition. 200âng Flag-ÎNT plasmid and 1000âng HA-C1 or HA-Ctrl plasmid were used in the co-transfection. Right panel: trimer bands were quantified, averaged, normalized, and compared by paired t-test (*P â¤ 0.001, N = 3).
	Figure 6. Effect of C1 on PKD2L1 trimerization and function.(A) Representative whole-cell current tracings from Xenopus oocytes expressing PKD2L1 (2L1) or PKD2L1/PKD1L3 (2L1+1L3), with or without C1 using the two-microelectrode voltage clamp technique under similar conditions as for Fig. 2. (B) Averaged currents elicited by extracellular 5âmM Ca2+ from oocytes expressing 2L1 or 2L1+1L3, with or without C1, and voltage clamped at â50âmV. Currents were averaged from different numbers of oocytes, as indicated, and normalized to that of WT PKD2L1 alone. (C) WB detection of PKD2L1 expression or dot blot detection of C1 expression in oocytes. (D) Representative currentâvoltage relationship curves obtained using a voltage ramp protocol, as indicated in Fig. 2C, before (7.5) and after (7.5+Ca) addition of 5âmM CaCl2. (E) Representative immunofluorescence data showing the oocyte plasma membrane expression of 2L1 and 2L1+1L3, with or without C1. (F) Surface expression was quantified and averaged from the indicated numbers of oocytes, and normalized to that of 2L1.
	Figure 7. Model illustrating how a PKD2L1 homo- or heterotetramerer can be formed.(A) A PKD2L1 (oval) homotetramer can be formed either through first homotrimerization followed by recruitment of a fourth subunit to the trimer by dimerization or through first homodimerization followed by recruitment of two subunits to form a trimer with a subunit in the dimer. Of notes, 1) the illustration is only to indicate trimeric and dimeric binding and does not intend to show how the pore region is organized; and 2) the drawn trimer does not intend to mean that there are two points of contact. (B) A PKD2L1 heterotetramer can be formed with a PKD1 homologue (square), eg, PKD1L3 or PKD1L1, through first trimerization of PKD2L1 and then recruitment of a PKD1 homologue, or reversely, through first heterodimerization between PKD2L1 and a PKD1 homologue, followed by recruitment of two PKD2L1 subunits to form a trimer with the existing PKD2L1 subunit.

References [+] :

Chen, Polycystin-L is a calcium-regulated cation channel permeable to calcium ions. 1999, Pubmed, Xenbase