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Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Polycystic kidney disease (PKD) is a genetic disorder characterized by fluid-filled cysts in the kidney and liver that ultimately leads to end-stage renal disease. Currently there is no globally approved therapy for PKD. The Notch signaling pathway regulates cellular processes such as proliferation and de-differentiation, which are cellular hallmarks of PKD. Thus we hypothesized that the Notch pathway plays a critical role in PKD. Evaluation of protein expression of Notch signaling components in kidneys of Autosomal Recessive PKD (ARPKD) and Autosomal Dominant PKD (ADPKD) mouse models and of ADPKD patients revealed that Notch pathway members, particularly Notch3, were consistently upregulated or activated in cyst-lining epithelial cells. Notch3 expression correlated with rapidly growing cysts and co-localized with the proliferation marker, PCNA. Importantly, Notch inhibition significantly decreased forskolin-induced Notch3 activation and proliferation of primary human ADPKD cells, and significantly reduced cyst formation and growth of human ADPKD cells cultured in collagen gels. Thus our data indicate that Notch3 is aberrantly activated and facilitates epithelial cell proliferation in PKD, and that inhibition of Notch signaling may prevent cyst formation and growth.

Primary cilia are small, antenna-like organelles that detect and transduce chemical and mechanical cues in the extracellular environment, regulating cell behavior and, in turn, tissue development and homeostasis. Primary cilia are assembled via intraflagellar transport (IFT), which traffics protein cargo bidirectionally along a microtubular axoneme. Ranging from 1 to 10 μm long, these organelles typically reach a characteristic length dependent on cell type, likely for optimum fulfillment of their specific roles. The importance of an optimal cilia length is underscored by the findings that perturbation of cilia length can be observed in a number of cilia-related diseases. Thus, elucidating mechanisms of cilia length regulation is important for understanding the pathobiology of ciliary diseases. Since cilia assembly/disassembly regulate cilia length, we review the roles of IFT in processes that affect cilia assembly/disassembly, including ciliary transport of structural and membrane proteins, ectocytosis, and tubulin posttranslational modification. Additionally, since the environment of a cell influences cilia length, we also review the various stimuli encountered by renal epithelia in healthy and diseased states that alter cilia length and IFT.

Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

Polycystin-1 transforms the cAMP growth-responsive phenotype of M-1 cells. Polycystic kidney disease (PKD) is characterized by the abnormal proliferation of tubular epithelial cells. It was recently shown that the growth of PKD cyst-lining cells is stimulated by cyclic adenosine monophosphate (cAMP), whereas the growth of normal human kidney cortex cells is inhibited. We have examined the effects of overexpressing the C-terminal cytosolic tail of mouse polycystin-1, as a membrane-targeted fusion protein, on cAMP-responsive cell proliferation in stably transfected M-1 cortical collecting duct cells. Two cell lines that express high levels of the polycystin-1 fusion protein and two control cell lines that do not express the fusion protein were tested. Growth of parental M-1 cells and the control cell lines was inhibited by 8-Br-cAMP and by a variety of cAMP agonists. In contrast, growth of the polycystin-1–expressing clones was stimulated by cAMP. Consistent with this, the protein kinase A (PKA) inhibitor H-89 caused either a positive or a negative growth effect depending on the primary response to cAMP. PD98059 blocked the cAMP stimulation of cell proliferation, indicating that the pathway is MEK1 dependent. Expression of the polycystin-1 C-terminal tail disrupts normal cellular signaling and transforms the stably transfected M-1 cells to an abnormal PKD cell proliferation phenotype.

Cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) are abnormally sensitive to ouabain, responding to physiological ouabain concentrations with enhanced proliferation and increased forskolin-induced transepithelial fluid secretion. This requires activation of the epidermal growth factor receptor (EGFR), Src kinase and the extracellular signal-regulated kinases MEK and ERK. Here, we have determined if the ADPKD phenotype obtained in mouse cortical collecting duct cells by stable overexpression of the C-terminal domain of polycystin-1 (PC-1 C-tail) also elicits the ADPKD-like response to ouabain in the cells. M-1 C20 cells expressing the PC-1 C-tail and M-1 C17 cells lacking expression of this construct were treated with physiological concentrations of ouabain, and cell proliferation, activation of the EGFR-Src-MEK-ERK pathway, forskolin-induced transepithelial Cl − secretion and the sensitivity of Na,K-ATPase to ouabain were explored. M-1 C20 cells responded to ouabain with increased cell proliferation and ERK phosphorylation. Ouabain also augmented forskolin-induced and cystic fibrosis transmembrane conductance regulator-mediated apical secretion of Cl − in M-1 C20 cells. These effects required activation of EGFR, Src and MEK. In contrast, ouabain had no significant effects on M-1 C17 cells. Interestingly, approximately 20 % of the Na,K-ATPase from M-1 C20 cells presented an abnormally increased sensitivity to ouabain. Overexpression of PC-1 C-tail in M-1 C20 cells is associated with an ouabain-sensitive phenotype and an increased ability of the cells to proliferate and secrete anions upon ouabain stimulation. This phenotype mimics the ouabain sensitivity of ADPKD cells and may help promote their cystogenic potential.

Renal activation of extracellular signal-regulated kinase in rats with autosomal-dominant polycystic kidney disease. Abnormal proliferation of renal tubule epithelial cells is a central factor in the biogenesis and sustained expansion of cysts in autosomal-dominant polycystic kidney disease (ADPKD). Recent evidence from in vitro studies of human cyst wall epithelial cells has implicated a role for the mitogen-activated protein (MAP) kinase pathway in this aberrant proliferation. To determine the extent to which this signaling pathway is involved in cyst pathogenesis in vivo, we measured the expression of select components of the MAP kinase cascade in Han:SPRD rats with ADPKD at an early stage of the disease. Kidneys of 8-week-old normal Han:SPRD rats (+/+) or rats heterozygous (Cy/+) for ADPKD were examined by Western blot analysis and immunohistochemistry to determine the expression of extracellular-regulated kinase (ERK), phosphorylated ERK (P-ERK), Raf-1 (MAPKKK), phosphorylated Raf-1 (P-Raf-1), B-Raf, Rap-1 and phosphorylated protein kinase A (P-PKA). P-ERK was expressed to a greater extent in Cy/+ kidneys (3.74 ± 1.07 fold) than in normal kidneys, whereas ERK abundance was not different. P-Raf-1 levels were higher in Cy/+ than in +/+ kidneys (1.53 ± 0.08 fold) consistent with upstream stimulation of receptor tyrosine kinase. B-Raf and Raf-1 abundances were greater in Cy/+ than in +/+ (1.74 ± 0.25 and 1.27 ± 0.08 fold, respectively). In Cy/+, immunohistochemistry showed increased P-ERK and B-Raf expression in the abnormal mural epithelial cells within cysts. These findings, together with the detection of P-PKA and the small G protein, Rap-1, in cyst epithelial cells, implicate a potential role for cyclic adenosine monophosphate (AMP) in the activation of ERK in ADPKD cells. We conclude that the MAP kinase pathway is activated to the level of ERK in the abnormal mural epithelial cells lining cysts in animals with a dominantly inherited type of polycystic kidney disease. We suggest that cAMP, acting through PKA, Rap-1 and B-Raf, may contribute to the activation of ERK in a way that complements receptor tyrosine kinase-mediated agonists in the promotion of cyst enlargement.

Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Polycystin-1 (PC1) is the membrane protein product of the PKD1 gene whose mutation is responsible for 85% of the cases of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is primarily characterized by the formation of renal cysts and potential kidney failure. PC1 is an atypical G protein-coupled receptor (GPCR) consisting of 11 transmembrane helices and an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal (NTF) and membrane-embedded C-terminal (CTF) fragments. Recently, signaling activation of the PC1 CTF was shown to be regulated by a stalk tethered agonist (TA), a distinct mechanism observed in the adhesion GPCR family. A novel allosteric activation pathway was elucidated for the PC1 CTF through a combination of Gaussian accelerated molecular dynamics (GaMD), mutagenesis and cellular signaling experiments. Here, we show that synthetic, soluble peptides with 7 to 21 residues derived from the stalk TA, in particular, peptides including the first 9 residues (p9), 17 residues (p17) and 21 residues (p21) exhibited the ability to re-activate signaling by a stalkless PC1 CTF mutant in cellular assays. To reveal molecular mechanisms of stalk peptide-mediated signaling activation, we have applied a novel Peptide GaMD (Pep-GaMD) algorithm to elucidate binding conformations of selected stalk peptide agonists p9, p17 and p21 to the stalkless PC1 CTF. The simulations revealed multiple specific binding regions of the stalk peptide agonists to the PC1 protein including an \"intermediate\" bound yet inactive state. Our Pep-GaMD simulation findings were consistent with the cellular assay experimental data. Binding of peptide agonists to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of the PC1 CTF signaling activation mechanism. Using sequence covariation analysis of PC1 homologs, we further showed that the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. Therefore, structural dynamic insights into the mechanisms of PC1 activation by stalk-derived peptide agonists have enabled an in-depth understanding of PC1 signaling. They will form a foundation for development of PC1 as a therapeutic target for the treatment of ADPKD.

Differential expression of Cux-1 and p21 in polycystic kidneys from Pkd1 null and cpk mice. Cux-1 is a murine homeodomain protein that functions as a cell cycle–dependent transcriptional repressor in proliferating cells. Targets of Cux-1 repression include the cyclin kinase inhibitors p21 and p27. In the kidney, Cux-1 is spatially and temporally regulated, and ectopic expression of Cux-1 in transgenic mice results in renal hyperplasia. Previously, we observed that Cux-1 is deregulated in cystic kidneys from cpk mice. Recent studies have suggested a role for the cyclin kinase inhibitor p21 in the development of polycystic kidney disease (PKD) in mice lacking PKD1. Since p21 is a target of transcriptional repression by Cux-1, we compared the expression of Cux-1 and p21 in kidneys from Pkd1 null and cpk mice by immunohistochemistry and Western blotting. We also evaluated apoptosis and the expression of the cyclin kinase inhibitor p27 in Pkd1 null and cpk mice by terminal deoxynucleotidal transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, immunohistochemistry, and Western blotting. In both early and late embryonic kidneys from Pkd1 null mice, Cux-1 was highly and ectopically expressed in normal-appearing tubule epithelium, interstitial cells, and in the epithelial cells lining the cysts, where it colocalized with proliferating cell nuclear antigen (PCNA). Increased Cux-1 expression in Pkd1 null kidneys was also associated with a decrease in p27 expression at late stages of cystogenesis. In cpk kidneys, Cux-1 was not up-regulated until late stages of cyst development. Moreover, in contrast to Pkd1 null kidneys, p21 and p27 were highly expressed in cpk kidneys. In late stages of cystogenesis, Cux-1 and p21 colocalized in cyst lining cells, which also showed a high incidence of apoptosis. These results suggest that cyst development in Pkd1 null mice and cpk mice proceeds through different mechanisms. In Pkd1 null mice, ectopic expression of Cux-1 is associated with increased cell proliferation. In contrast, in cpk mice, ectopic expression of Cux-1 is associated with apoptosis.