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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).

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.

SummaryDuring the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) The AHCY family AHCY S-adenosyl-L-homocystein (SAH) hydrolase (AHCY) is a ubiquitous, tetrameric enzyme that catalyzes the reversible hydrolysis of SAH to adenosine and L-homocysteine. SAH is formed as a by-product of S-adenosyl-L-methionine (SAM) through transmethylation reactions. The hydrolysis of SAH is required to maintain low cellular concentrations of SAH, which is a product inhibitor of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions.(1-4) Inhibition of AHCY results in the intracellular accumulation of SAH, causing a significant increase in the intracellular SAH/SAM ratio and the subsequent inhibition of SAM-dependent methylations.( 5 -8) SAM is the major methyl donor for delivery of methyl groups to DNA, RNA, proteins and cellular metabolites in eukaryotes and SAH hydrolysis is the only source of homocysteine in mammals.(9) AHCY has become an attractive target for design of broad-spectrum antiviral agents because of the inhibitory effects of elevated intracellular SAH concentrations on viral mRNA cap-methylating enzymes. (10,11) In addition to these antiviral effects, AHCY inhibitors have also been attributed antiparasitic, (12,13) anti-arthritic (14) and immunosuppressive (15) effects.The importance of AHCY for mammalian survival is suggested by the fact that a chromosomal deletion that includes the gene encoding AHCY causes embryonic lethality in mice.(16) Elevated homocysteine levels have been reported as a risk factor for dementia and Alzheimer's disease, (17) and as a possible risk marker for vascular disease. (18,19) Human AHCY deficiency, such as in hypermethionemia, results in severe biochemical abnormalities, including plasma elevations of 150-fold in SAH, 30-fold in SAM and 12-fold in methionine. (20,21) So far, three mutations in the AHCY gene have been found to reduce the activity of the AHCY enzyme, resulting in the signs and symptoms of hypermethionemia. (22,23) AHCY orthologues have been identified in many species, including bacteria, nematodes, yeast, plants, insects and vertebrates. In contrast, the AHCY-like (AHCYL) family members AHCYL1 (also termed IRBIT, the inositol 1,4, 5-trisphosphate (IP 3 ) receptor (IP 3 R) binding protein released by IP 3 ) and AHCYL2 (KIAA0828 in human) are only predicted in segmented multicellular organisms, like vertebrates (eg Takifugu rubripes (fugu fish), Danio rerio (zebrafish), Xen...

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