Structural and molecular basis of the assembly of the TRPP2/PKD1 complex

There is functional evidence that polycystin-2 (TRPP2) interacts with other members of the transient receptor potential family, including TRPC1 and TRPV4. Here we have used atomic force microscopy to study the structure of the TRPP2 homomer and the interaction between TRPP2 and TRPC1. The molecular volumes of both Myc-tagged TRPP2 and V5-tagged TRPC1 isolated from singly transfected tsA 201 cells indicated that they assembled as homotetramers. The molecular volume of the protein isolated from cells expressing both TRPP2 and TRPC1 was intermediate between the volumes of the two homomers, suggesting that a heteromer was being formed. The distribution of angles between pairs of anti-Myc antibodies bound to TRPP2 particles had a large peak close to 90°and a smaller peak close to 180°, consistent with the assembly of TRPP2 as a homotetramer. In contrast, the corresponding angle distributions for decoration of the TRPP2-TRPC1 heteromer by either anti-Myc or anti-V5 antibodies had predominant peaks close to 180°. This decoration pattern indicates a TRPP2:TRPC1 subunit stoichiometry of 2:2 and an alternating subunit arrangement.Autosomal dominant polycystic kidney disease is one of the commonest inherited human disorders (reviewed in Ref. 1). It has a population prevalence of over 1:1,000 in all ethnic groups and is a leading cause of end stage renal failure. Autosomal dominant polycystic kidney disease is characterized by the progressive loss of normal renal parenchyma secondary to the development of multiple fluid-filled cysts derived from renal tubular epithelial cells. It is caused by mutations in two genes, PKD1 and PKD2, whose protein products, polycystin-1 (2, 3) and polycystin-2 (or TRPP2) (4) form a Ca 2ϩ -permeable ion channel complex (5). This complex transduces extracellular mechanical stimuli via the renal primary cilium (6) and regulates multiple intracellular Ca 2ϩ -sensitive signaling pathways (5, 7). TRPP2 also appears to have a role, independent of polycystin-1, in regulating Ca 2ϩ efflux from the endoplasmic reticulum (8, 9). In addition to its interaction with polycystin-1, TRPP2 is known to interact with other members of the TRP 4 superfamily, such as TRPC1 (10) and TRPV4 (11), raising the question of the architecture of these heteromeric complexes.TRP channel complexes have been assumed to be tetramers, initially on the basis of the resemblance of the primary structure of the TRP channel subunits to that of the Shaker K ϩ channel, which is known to be tetrameric (12). Moreover, a variety of structural and functional techniques have been used to demonstrate a tetrameric structure for a number of TRP channel family members, including TRPC1 (13), TRPC3 (14), TRPV1 (15, 16), TRPV5 and TRPV6 (17), and TRPM2 (18). Intriguingly, data have recently been presented indicating that TRPP2 exists in the plasma membrane as a trimer, which is then able to interact with polycystin-1 to form a heteromer with a 3:1 stoichiometry (19). It is puzzling that TRPP2 appears to behave differently from all other TRP channel...

“…This finding directly contradicts the conclusion of a recent report that TRPP2 in the plasma membrane is a trimer (19). The evidence presented in the previous report is 3-fold.…”

Section: Discussioncontrasting

confidence: 57%

mentioning

confidence: 99%

The Transient Receptor Potential Channels TRPP2 and TRPC1 Form a Heterotetramer with a 2:2 Stoichiometry and an Alternating Subunit Arrangement

Kobori

Smith

Sandford

et al. 2009

“…However an atomic resolution structure for a full-length TRP channel is not yet available. Isolated domains of TRP channels have been solved by x-ray crystallography or NMR, including the ␣-kinase domain of TRPM7 (10), the ankyrin repeat domains from the TRPV subfamily of proteins (11)(12)(13)(14)(15), the C-terminal cytoplasmic coiled-coil domains of TRPM7 and TRPP2 (16,17), and the EF hand domain of TRPP2 (18,19). These partial atomic structures are useful for understanding channel regulation/modulation and enhance the interpretation of moderate resolution fulllength TRP channel structures (20).…”

Section: The Transient Receptor Potential (Trp)mentioning

confidence: 99%

Molecular Architecture and Subunit Organization of TRPA1 Ion Channel Revealed by Electron Microscopy

Cvetkov

Huynh

Cohen

et al. 2011

111

117

Background:The TRPA1 ion channel plays critical roles in pain and inflammatory processes. Results: A TRPA1 structure is presented at 16-Å resolution with docked molecular models. Conclusion: Channel activation could involve conformation changes resulting from ligands binding in a pocket proposed to bridge adjacent subunits. Significance: Structural-functional understanding of TRPA1 is important for resolving fundamental pain and inflammatory mechanisms.

“…There are two Ca 2ϩ -binding sites (aa 680 -796) arranged in a typical and an atypical EF-hand motif, which could be involved in a Ca 2ϩ -mediated regulation of TRPP2 (6). An endoplasmic reticulum (ER) retention signal (aa 787-820) (7) and a coiled-coil domain (aa 839 -919), responsible for homo-and heterodimerization (8,9), are also present. Recently, it was reported that this coiled-coil domain was responsible for formation of a TRPP2 trimer that interacts with PKD1 in the plasma membrane (9).…”

mentioning

confidence: 99%

“…An endoplasmic reticulum (ER) retention signal (aa 787-820) (7) and a coiled-coil domain (aa 839 -919), responsible for homo-and heterodimerization (8,9), are also present. Recently, it was reported that this coiled-coil domain was responsible for formation of a TRPP2 trimer that interacts with PKD1 in the plasma membrane (9). * This work was supported in part by Grant GOA/09/012 from the Concerted Actions of the K.U.…”

mentioning

confidence: 99%

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Sammels

Devogelaere

Mekahli

et al. 2010

113

117

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca 2؉ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP 3 R), an intracellular Ca 2؉ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP 3 R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2 ؊/؊ mouse renal epithelial cells using an adenoviral expression system. signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease. Autosomal dominant polycystic kidney disease (ADPKD)4 is an inherited human disorder that affects more than six million people worldwide and is the most common monogenic cause of kidney failure in humans (1). ADPKD results in end-stage renal disease in ϳ50% of the affected individuals by the age of 60. ADPKD arises as a consequence of mutations of two genes PKD1 and PKD2, encoding integral membrane proteins polycystin-1 (PKD1, ϳ460 kDa) and polycystin-2 (TRPP2, ϳ110 kDa), respectively. Most mutations identified in affected families appear to truncate and (or) inactivate either of both proteins (2-5). Mutations in PKD1 account for the vast majority (ϳ85%) of patients with ADPKD and are associated with a more severe clinical presentation and earlier onset of end-stage renal disease than the PKD2 phenotype (4). However, in all other aspects, PKD1 and PKD2 mutations produce virtually indistinguishable disease manifestations, indicating that the two proteins might function in a common signaling pathway involved in maintaining the terminally differentiated state of renal epithelial cells.TRPP2 is a 968-amino acid (aa) protein with six predicted transmembrane domains and is highly conserved among multicellular organisms and widely expressed in various tissues (2). Structural analyses indicate that TRPP2 contains several functional domains in its C-terminal tail. There are two Ca 2ϩ -binding sites (aa 680 -796) arranged in a typical and an atypical EF-hand motif, which could be involved in a Ca 2ϩ -mediated regulation of TRPP2 (6). An endoplasmic reticulum (ER) retention signal (aa 787-820) (7) and a coiled-coil domain (aa 839 -919), responsible for homo-and heterodimerization (8,9), are also present. Recently, it was reported that this coiled-coil domain was responsible for formation of a TRPP2 trimer that interacts with PKD1 in the plasma membrane (9).