Structural and molecular basis of the assembly of the TRPP2/PKD1 complex Yong Yua, Maximilian H. Ulbrichb, Ming-Hui Lia, Zafir Buraeia, Xing-Zhen Chend, Albert C. M. Onge, Liang Tonga, Ehud Y. Isacoffb,c, and Jian Yanga,1 aDepartment of Biological Sciences, Columbia University, New York, NY 10027; bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; cMaterial Science and Physical Bioscience Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; dMembrane Protein Research Group, Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7; and eKidney Genetics Group, Academic Unit of Nephrology, Sheffield Kidney Institute, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom Edited by Christopher Miller, Brandeis University, Waltham, MA, and approved May 20, 2009 (received for review April 2, 2009) Mutations in PKD1 and TRPP2 account for nearly all cases of autoso- structural basis of the homomeric assembly of a functionally mal dominant polycystic kidney disease (ADPKD). These 2 proteins important TRPP2 coiled-coil domain, uncovers the crucial role form a receptor/ion channel complex on the cell surface. Using a of this domain in the assembly of the TRPP2/PKD1 complex, and combination of biochemistry, crystallography, and a single-molecule reveals an unexpected subunit stoichiometry of this complex. method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 Results TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C Subunit Stoichiometry of the TRPP2 Homomultimer Expressed in a Cell terminus of TRPP2 is critical for the formation of this complex. This Line. To study the molecular mechanism of the assembly of the coiled-coil domain forms a homotrimer, in both solution and crystal TRPP2/PKD1 complex, we first examined the stoichiometry of the structure, and binds to a single coiled-coil domain in the C terminus TRPP2 complex in the absence of PKD1. TRPP2 purified from a of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer HEK 293T cell line stably expressing HA-TRPP2 [TRPP2 tagged with hemagglutinin (HA) on the N terminus] was analyzed by blue abolish the assembly of both the full-length TRPP2 trimer and the native PAGE (BN-PAGE). Undenatured and unreduced TRPP2 TRPP2/PKD1 complex and diminish the surface expression of both protein migrated as a complex with an apparent molecular mass of proteins. These results have significant implications for the assembly, Ϸ850 kDa (Fig. 1A, lane 1). Denatured and reduced protein regulation, and function of the TRPP2/PKD1 complex and the patho- migrated as a complex with an apparent molecular mass of Ϸ280 genic mechanism of some ADPKD-producing mutations. kDa (Fig. 1A, lane 2). In contrast, denatured but unreduced protein showed 3 products, with an apparent molecular mass of Ϸ280, autosomal dominant polycystic kidney disease ͉ single-molecule imaging ͉ Ϸ600, and Ϸ850 kDa (Fig. 1A, lane 3). The relative size of the 3 stoichiometry ͉ transient receptor potential channel ͉ X-ray crystallography products suggests that they correspond to the TRPP2 monomer, dimer, and trimer. The apparent molecular mass of these products utosomal dominant polycystic kidney disease (ADPKD), is much larger than their predicted molecular mass (110, 220, and Aone of the most common genetic diseases in humans, is 330 kDa, respectively). Glycosylation, detergent binding, and/or caused by mutations in PKD1 or TRPP2 (1–3). PKD1 (also anomalous mobility in BN-PAGE probably account for their much known as polycystin-1 or PC1) is a 4,302-aa, 465-kDa integral higher observed molecular mass. membrane protein containing 11 putative transmembrane re- We next used chemical cross-linking to further determine the subunit stoichiometry of the TRPP2 homomeric complex. Treat- gions (4) (Fig. S1). Its large extracellular N terminus contains a ment of the lysate of the HA-TRPP2-expressing cells with the number of well-recognized repeats and domains, some of which amine-reactive cross-linker DST resulted in 3 products, with an are known to interact with extracellular matrix proteins. The apparent molecular mass of 120, 260, and 400 kDa (Fig. 1B), which short intracellular C terminus contains a G protein activation likely correspond to the TRPP2 monomer, dimer, and trimer (the site. Thus, PKD1 is generally thought to function as a cell surface largest band is unlikely to be the tetramer because the extrapolated receptor for extracellular ligands and matrix proteins that cou- molecular mass of the tetramer would be Ϸ520 kDa). These and the ples extracellular stimuli, including mechanical stimuli, to intra- preceding results indicate that the prevalent form of TRPP2 in the cellular signaling (1, 4–6). TRPP2 (also known as PKD2, poly- cell lysate is a trimer. cystin-2 or PC2) is a member of the transient receptor potential We also produced 2 other HEK 293T stable lines, expressing (TRP) channel family (7). TRPP2 is a 968-aa, 110-kDa integral either FLAG-PKD1 (PKD1 tagged with FLAG on the N terminus) membrane protein with 6 putative transmembrane segments and or FLAG-PKD1 plus HA-TRPP2. Because of technical challenges, a pore-forming loop (7) (Fig. S1) and forms a Ca2ϩ-permeable our attempts to cross-link TRPP2 and PKD1 produced inconclusive nonselective cation channel (8–10). results. How mutations in PKD1 and TRPP2 lead to ADPKD is unclear. The 2 proteins likely participate in the same molecular and cellular Subunit Stoichiometry of the TRPP2/PKD1 Heteromeric and TRPP2 processes in some kidney cells, because their mutations produce Homomeric Complex in the Membrane of Live Cells. Next, we used a similar pathological manifestations. Although both proteins are single-molecule optical approach (22) to determine the stoichiom- widely distributed, they show high levels of expression in kidney cells (1, 5–7), where they are present on the plasma membrane and Author contributions: Y.Y., M.H.U., M.-H.L., Z.B., X.-Z.C., A.C.M.O., L.T., E.Y.I., and J.Y. in the Golgi and endoplasmic reticulum (1, 4–6, 11–13). They also designed research; Y.Y., M.H.U., M.-H.L., and Z.B. performed research; Y.Y., M.H.U., Z.B., colocalize in the primary cilia of kidney epithelium (14). The 2 and J.Y. analyzed data; and Y.Y., M.H.U., E.Y.I., and J.Y. wrote the paper. proteins associate physically (15–19) and form functional complexes The authors declare no conflict of interest. (18, 20, 21). However, the subunit composition of this heteromeric This article is a PNAS Direct Submission. complex and the molecular mechanism underlying its assembly are Data deposition: The atomic coordinates and structure factors have been deposited in unknown. Protein Data Bank, www.pdb.org (PDB ID codes 3HRN and 3HRO). In this work we investigated these issues by using a multifac- 1To whom correspondence should be addressed. E-mail: [email protected]. eted approach that includes biochemistry, X-ray crystallography, This article contains supporting information online at www.pnas.org/cgi/content/full/ and single-molecule optical imaging. Our study provides the 0903684106/DCSupplemental. 11558–11563 ͉ PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903684106 Downloaded by guest on September 24, 2021 Fig. 1. Subunit stoichiometry of full-length homomeric TRPP2 and heteromeric TRPP2/PKD1 complexes. (A) Western blot analysis after BN-PAGE of purified HA-TRPP2. In lanes 2 and 3, the sample was incubated at 70 °C for 10 min with 2% SDS plus 100 mM DTT or with 2% SDS. TRPP2 was detected with an anti-HA antibody, and putative TRPP2 monomer, dimer, and trimer are indicated. (B) Western blot analysis after SDS/PAGE showing the cross-linking products of TRPP2. Cross-linking was carried out with 1 mM DST. The putative subunit composition of the bands is indicated. (C and F) TIRF images of EGFP and mCherry fluorescence from an oocyte expressing the BIOCHEMISTRY indicated constructs, showing spots that exhibited EGFP and mCherry dual fluorescence and were immobile (circles). Other spots do not fit analysis criteria. (Scale bars: 2 ␮m.) (D) Time course of photobleaching of 2 representative PKD1-mCherry and TRPP2-EGFP dual-fluorescence spots, showing 3 EGFP bleaching steps (arrows). mCherry and EGFP excitation is indicated by red and green bars, respectively. (E) Distribution of observed EGFP bleaching steps (green bars) for PKD1-mCherry and TRPP2-EGFP dual-fluorescence spots compared with calculated distribution based on 83% of EGFPs being fluorescent (white bars). No events with 5 or more bleaching steps were observed. (G) Time course of photobleaching of 2 representative PKD1-EGFP and TRPP2-mCherry dual-fluorescence spots showing 1 EGFP bleaching step (arrow). (H) Distribution of EGFP bleaching steps for PKD1-EGFP and TRPP2-mCherry dual-fluorescence spots. etry of the TRPP2/PKD1 complex and the homomeric TRPP2 to a binomial distribution that assumes that each spot contains 4 complex in the membrane of live cells. This method uses total EGFPs (Fig. S2). In the complementary experiment in oocytes internal reflection fluorescence (TIRF) microscopy to visualize expressing PKD1-EGFP and TRPP2-mCherry (Fig. 1F), most single, GFP-tagged proteins exclusively on the plasma membrane of dual-fluorescence spots showed only 1 EGFP bleaching step (Fig. Xenopus oocytes. Measurements were made simultaneously for 1 G and H). A small minority of spots showed 2 EGFP bleaching many complexes at low levels of protein expression, where the steps (Fig. 1H), probably because they contained 2 complexes. formation of non-native, high-order complexes and aggregates is mCherry showed a gradual decrease in fluorescence, indicating the avoided. The number of GFP photobleaching steps was used to presence of several subunits (Fig.