Identification and functional characterization of an N-terminal oligomerization domain for polycystin-2*

Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function

Control of PTH secretion by the TRPC1 ion channel

Familial Hypocalciuric Hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia and in some cases inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in CaSR encoding the Ca2+ sensing receptor (CaSR), a G protein coupled receptor (GPCR) and GNA11 encoding G protein subunit alpha 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels mimicking human FHH. Ex vivo and in vitro studies reveal that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTG) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacts with both the N- and C-termini of TRPC1 and enhances CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

<p>Abstract</p> <p>Background</p> <p>Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes <it>PKD1 </it>and <it>PKD2 </it>account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of <it>PKD2</it>. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.</p> <p>Methods</p> <p>Here we utilized different kidney cell-lines expressing wild-type and mutant <it>PKD2 </it>as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.</p> <p>Results</p> <p>Surprisingly, over-expression of wild-type <it>PKD2 </it>in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated <it>PKD2 </it>augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57^KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.</p> <p>Conclusion</p> <p>Our results indicate the probable involvement of p57^KIP2on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.</p

Function and regulation of TRPP2 at the plasma membrane

The vast majority (∼99%) of all known cases of autosomal dominant polycystic kidney disease (ADPKD) are caused by naturally occurring mutations in two separate, but genetically interacting, loci, pkd1 and pkd2. pkd1 encodes a large multispanning membrane protein (PKD1) of unknown function, while pkd2 encodes a protein (TRPP2, polycystin-2, or PKD2) of the transient receptor potential (TRP) superfamily of ion channels. Biochemical, functional, and genetic studies support a model in which PKD1 physically interacts with TRPP2 to form an ion channel complex that conveys extracellular stimuli to ionic currents. However, the molecular identity of these extracellular stimuli remains elusive. Functional studies in cell culture show that TRPP2 can be activated in response to mechanical cues (fluid shear stress) and/or receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) activation at the cell surface. Recent genetic studies in Chlamydomonas reinhardtii show that CrPKD2 functions in a pathway linking cell-cell adhesion and Ca2+ signaling. The mode of activation depends on protein-protein interactions with other channel subunits and auxiliary proteins. Therefore, understanding the mechanisms underlying the molecular makeup of TRPP2-containing complexes is critical in delineating the mechanisms of TRPP2 activation and, most importantly, the mechanisms by which naturally occurring mutations in pkd1 or pkd2 lead not only to ADPKD, but also to other defects reported in model organisms lacking functional TRPP2. This review focuses on the molecular assembly, function, and regulation of TRPP2 as a cell surface cation channel and discusses its potential role in Ca2+ signaling and ADPKD pathophysiology

Insights into the Regulation of Ciliary Disassembly

The primary cilium, an antenna-like structure that protrudes out from the cell surface, is present in most cell types. It is a microtubule-based organelle that serves as a mega-signaling center and is important for sensing biochemical and mechanical signals to carry out various cellular processes such as proliferation, migration, differentiation, and many others. At any given time, cilia length is determined by a dynamic balance of cilia assembly and disassembly processes. Abnormally short or long cilia can cause a plethora of human diseases commonly referred to as ciliopathies, including, but not limited to, skeletal malformations, obesity, autosomal dominant polycystic kidney disease, retinal degeneration, and bardet-biedl syndrome. While the process of cilia assembly is studied extensively, the process of cilia disassembly and its biological role(s) are less well understood. This review discusses current knowledge on ciliary disassembly and how different cellular processes and molecular signals converge to carry out this process. This information will help us understand how the process of ciliary disassembly is regulated, identify the key steps that need further investigation, and possibly design therapeutic targets for a subset of ciliopathies that are causally linked to defective ciliary disassembly

Cellular Mechanisms of Ciliary Length Control

Cilia and flagella are evolutionarily conserved, membrane-bound, microtubule-based organelles on the surface of most eukaryotic cells. They play important roles in coordinating a variety of signaling pathways during growth, development, cell mobility, and tissue homeostasis. Defects in ciliary structure or function are associated with multiple human disorders called ciliopathies. These diseases affect diverse tissues, including, but not limited to the eyes, kidneys, brain, and lungs. Many processes must be coordinated simultaneously in order to initiate ciliogenesis. These include cell cycle, vesicular trafficking, and axonemal extension. Centrioles play a central role in both cell cycle progression and ciliogenesis, making the transition between basal bodies and mitotic spindle organizers integral to both processes. The maturation of centrioles involves a functional shift from cell division toward cilium nucleation which takes place concurrently with its migration and fusion to the plasma membrane. Several proteinaceous structures of the distal appendages in mother centrioles are required for this docking process. Ciliary assembly and maintenance requires a precise balance between two indispensable processes; so called assembly and disassembly. The interplay between them determines the length of the resulting cilia. These processes require a highly conserved transport system to provide the necessary substances at the tips of the cilia and to recycle ciliary turnover products to the base using a based microtubule intraflagellar transport (IFT) system. In this review; we discuss the stages of ciliogenesis as well as mechanisms controlling the lengths of assembled cilia

Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2

PKD1 and PKD2 are two recently identified genes that are responsible for the vast majority of autosomal polycystic kidney disease, a common inherited disease that causes progressive renal failure. PKD1 encodes polycystin, a large glycoprotein that contains several extracellular motifs indicative of a role in cell–cell or cell–matrix interactions, and the PKD2 encodes a protein with homology to a voltage-activated calcium channel and to PKD1. It is currently unknown how mutations of either protein functionally cause autosomal polycystic kidney disease. We show that PKD1 and PKD2 interact through their C-terminal cytoplasmic tails. This interaction resulted in an up-regulation of PKD1 but not PKD2. Furthermore, the cytoplasmic tail of PKD2 but not PKD1 formed homodimers through a coiled–coil domain distinct from the region required for interaction with PKD1. These interactions suggest that PKD1 and PKD2 may function through a common signaling pathway that is necessary for normal tubulogenesis and that PKD1 may require the presence of PKD2 for stable expression

Specific association of the gene product of PKD2 with the TRPC1 channel

The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell–cell or cell–matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca(2+) channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca(2+) stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2–TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca(2+) entry in response to G protein-coupled receptor activation and/or store depletion

PKD2 Functions as an Epidermal Growth Factor-Activated Plasma Membrane Channel

PKD2, or polycystin 2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, belongs to the transient receptor potential channel superfamily and has been shown to function as a nonselective cation channel in the plasma membrane. However, the mechanism of PKD2 activation remains elusive. We show that PKD2 overexpression increases epidermal growth factor (EGF)-induced inward currents in LLC-PK(1) kidney epithelial cells, while the knockdown of endogenous PKD2 by RNA interference or the expression of a pathogenic missense variant, PKD2-D511V, blunts the EGF-induced response. Pharmacological experiments indicate that the EGF-induced activation of PKD2 occurs independently of store depletion but requires the activity of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Pipette infusion of purified phosphatidylinositol-4,5-bisphosphate (PIP(2)) suppresses the PKD2-mediated effect on EGF-induced conductance, while pipette infusion of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) does not have any effect on this conductance. Overexpression of type Iα phosphatidylinositol-4-phosphate 5-kinase [PIP(5)Kα], which catalyzes the formation of PIP(2), suppresses EGF-induced currents. Biochemical experiments show that PKD2 physically interacts with PLC-γ2 and EGF receptor (EGFR) in transfected HEK293T cells and colocalizes with EGFR and PIP(2) in the primary cilium of LLC-PK(1) cells. We propose that plasma membrane PKD2 is under negative regulation by PIP(2). EGF may reduce the threshold of PKD2 activation by mechanical and other stimuli by releasing it from PIP(2)-mediated inhibition

Cellular Activation Triggered by the Autosomal Dominant Polycystic Kidney Disease Gene Product PKD2

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) ɛ, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC α and PKC ɛ, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts