Unraveling the role of polycystin-2/inositol 1,4,5-trisphosphate receptor interaction in Ca²⁺ signaling

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) arises as a consequence of mutations of the genes PKD1 and PKD2, encoding respectively the integral membrane proteins polycystin-1 and polycystin-2 (TRPP2), resulting in a disturbance in intracellular Ca²⁺ signaling. Previously we investigated the interaction between TRPP2 and the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum (ER). We identified the molecular determinants of this interaction and observed an enhanced IP₃-induced Ca²+ release (IICR). Since we found that TRPP2 strongly bound to a cluster of positively charged amino acids in the N-terminal ligand-binding domain (LBD) of the IP₃R, we now investigated whether TRPP2 would interfere with the binding of IP₃ to the IP₃R. In in vitro experiments we observed that TRPP2 partially inhibited the binding of IP₃ to the LBD of the IP₃R with an IC50 of ~350 nM. The suppressor domain, i.e. the N-terminal 225 amino acids of the LBD of the IP₃R, mediated this inhibitory effect of TRPP2 on IP₃ binding. The observation that the interaction between the IP3R and TRPP2 decreased IP3 binding is in apparent contrast to the increased IICR. The data can be explained however by a subsequent activation of Ca²⁺-induced Ca²⁺ release (CICR) via TRPP2. Implications of this mechanism for cellular Ca²⁺ signaling are discussed in this addendum.

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The inherited human disorder ADPKD affects more than 1 in 1,000 live births and is the most common monogenic cause of kidney failure in man.1 It is characterized by the progressive formation and enlargement of renal cysts, typically leading to chronic renal failure by late middle age.2 In most cases, the disease arises as a consequence of mutations in the PKD1 or PKD2 genes, which respectively encode the proteins polycystin-1 and TRPP2.1,3 TRPP2 is a 968-amino-acid (aa) protein with six predicted transmembrane domains, highly conserved among multicellular organisms and widely expressed in various tissues.4 TRPP2 has been implicated in diverse functions depending on its subcellular localization. TRPP2 has been detected (1) in the plasma membrane, where it is supposed to form a receptor-operated, non-selective cation channel,5 (2) in the primary cilium, where it could act as a mechanosensitive channel, possibly in association with other TRP-family members,6 (3) in the ER, where it is proposed to function as an intracellular Ca²⁺-release channel,7 and (4) also in centrosomes and in mitotic spindles of dividing cells (reviewed in refs. 8–10). However, the predominant subcellular localization of TRPP2 is in the ER, as shown by its sensitivity to endoglycosidase H, immunofluorescence experiments and its co-localization and co-distribution with ER-resident proteins.7,11 Previously we investigated the interaction between TRPP2 and the IP₃R, an ubiquitous intracellular Ca²⁺-release channel.12 We observed a strong interaction between TRPP2 and the IP₃R and identified a conserved positively charged cluster in the N-terminal LBD of the IP₃R and an acidic cluster located at the end of the ER-retention signal in the C-terminal tail of TRPP2 as being crucial for their interaction. When full-length TRPP2 was re-introduced in TRPP2^-/- mouse renal epithelial cells, there was a clear potentiation of agonist-induced intracellular Ca²⁺ release in intact cells and of IICR in permeabilized cells. Further analysis using pathological mutants of TRPP2 and competing peptides revealed that this effect on IICR was dependent on the TRPP2-channel function but in addition required interaction with the IP₃R.12

Since we found that TRPP2 interacted with the LBD of the IP₃R, we investigated whether TRPP2 was able to affect the IP₃-binding properties of a HIS-fusion protein of the LBD of IP₃R1 (LBD-HIS). The LBD consists of an IP₃-binding core (aa 226–581) and a suppressor domain (aa 1–225).13 A [³H]IP₃-binding assay14 was performed with purified LBD-HIS (aa 1–581) and with LBD-HIS Δ1–225 (aa 226–581) (described in ref. 15). In the presence of increasing concentrations of a GST-fusion protein of the C-terminal tail of murine TRPP2 (GST-TRPP2-CT, aa 679–966) we observed inhibition of the binding of IP₃ to the complete LBD, with an IC₅₀ of 350 nM (Fig. 1). This inhibition was partial and amounted to maximally 60% of the total binding. Deletion of the N-terminal 225 amino acids of the LBD, including the positive cluster that interacts with TRPP2,12 completely abolished the effect of GST-TRPP2-CT on IP₃ binding (Fig. 1). This confirms the role of the suppressor domain of the IP₃R as the major determinant for binding to the C-terminal tail of TRPP2.

The attenuation of IP₃ binding did however not inhibit Ca²⁺ release in functional assays of agonist-induced Ca²⁺ release or IICR with the full-length proteins. In contrast, we observed an enhanced Ca²⁺ release in the presence of TRPP2, which could be ascribed to the activation of TRPP2 via CICR. Moreover, in the functional assays we observed no difference in Ca²⁺ release between cells treated with a control adenovirus or cells expressing the pathological dead-channel mutant of TRPP2, D509V, which can still bind to the IP₃R.12 The inhibitory effect of TRPP2 on IP₃ binding in in vitro experiments was thus not observed in an intact cell context. Several arguments can explain this observation.

First, the inhibition of IP₃ binding was only partial. Apparently a conformational change induced by an allosteric interaction with the suppressor domain attenuates IP₃ binding to the IP₃-binding core but does not preclude subsequent IICR. This behavior is reminiscent to the interaction of the suppressor domain with calmodulin,16 which modulates but not by itself inhibits IICR. Secondly, it is possible that our functional assays, measuring global Ca²⁺ signals in a whole population of cells, did not have sufficient resolution to measure probably relatively small effects caused by TRPP2-mediated attenuation of IP₃ binding to the IP₃R. Thirdly, it is possible that interaction with TRPP2 resulted in modulation of the IP₃ response from a more graded into an all-or-none Ca²⁺ response. At low doses of IP₃ TRPP2 would inhibit the binding of IP₃ to the IP₃R and thus reduce IICR, thereby affecting the elementary events produced by IP₃Rs (called Ca²⁺ puffs).17,18 At higher doses of IP₃ this inhibition will be overcome and will induce IICR, which further activates CICR via the TRPP2 channel itself, resulting in an increased global Ca²⁺ signal. Discrimination of graded versus all-or-none Ca²⁺ response is difficult to achieve by measuring global Ca²⁺ changes at relatively high [IP₃] as was done by Sammels et al.12 Measuring elementary Ca²⁺ events in single cells could possibly elucidate this issue.

We can only speculate on the cellular significance of such a mechanism. All-ornone Ca²⁺ signals would be expected to be more restricted in time and space and to be localized in the immediate environment of the IP₃R/TRPP2 complexes. In this way the ER localization of TRPP2 would not result in Ca²⁺ increase at low or basal [IP₃] but would only result in local rises in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) evoked upon appropriate cell stimulation. It is conceivable that in the direct vicinity of the IP₃R/TRPP2 protein complex other signaling proteins are localized as adenylyl cyclases or phosphodiesterases (Fig. 2). In that respect, it is important to note that the Ca²⁺-dependent adenylyl cyclase VI was already found to be associated to IP₃Rs.19 As a result, our model (Fig. 2) proposes that the specificity of downstream Ca²⁺-dependent effects is further increased, e.g., by modulation of the [cAMP]. This can be relevant for the pathology of ADPKD, since increased levels of cAMP are a common finding in the kidneys of ADPKD animal models.20

We conclude that a signaling complex involving TRPP2 and the IP₃R is important for modulating intracellular Ca²⁺ signaling. Disturbance of this interaction, which occurs in pathologically relevant mutants of TRPP2, will lead to altered intracellular Ca²⁺ homeostasis and might contribute to the development of ADPKD caused by loss-of-function mutations in TRPP2. We found that TRPP2 activation via CICR required an initial rise in [Ca²⁺]_cyt via IICR and an interaction with the IP₃R. We observed that TRPP2 could inhibit the binding of IP₃ to its receptor in vitro. Taken together, these properties could favour the specificity of intracellular Ca²⁺ signaling evoked by ER-localized TRPP2.

Abbreviations

AC-VI	=	adenylyl cyclase type 6
ADPKD	=	autosomal dominant polycystic kidney disease
[Ca2+]cyt	=	free cytosolic Ca2+ concentration
CICR	=	Ca2+-induced Ca2+ release
ER	=	endoplasmic reticulum
IICR	=	inositol 1,4,5-trisphosphate-induced Ca2+ release
IP3	=	inositol 1,4,5-trisphosphate
IP3R	=	inositol 1,4,5-trisphosphate receptor
LBD	=	ligand-binding domain
PDE	=	phosphodiesterase
TRPP2	=	polycystin-2

Figures and Tables

Figure 1 The effect of TRPP2-CT on IP₃ binding to the IP₃R LBD. Specific binding of 1.5 nm [³H]IP₃ to recombinant HIS-fusion proteins of the LBD of the IP₃R, consisting of the suppressor domain and the IP₃-binding core (red) and of the IP₃-binding core, which lacks the suppressor domain, (blue) in the presence of increasing concentrations of recombinant GST-fusion protein of the C-terminal tail of TRPP2. The mean ± S.E.M. of three independent experiments is shown.

Figure 2 Proposed model. The binding of TRPP2 to the suppressor domain of the IP₃R partially inhibits IP₃ binding in vitro (dashed line), thus possibly suppressing Ca²⁺ release events at very low [IP₃]. Upon appropriate cell stimulation, when a sufficiently high [IP₃] is produced, the IP₃R is activated leading to a local [Ca²⁺]_cyt rise that subsequently activates the TRPP2 channel as a CICR channel. We suggest that a signaling microdomain, where TRPP2 interacts with the IP₃R through the charged residues, is required to facilitate CICR by TRPP2. In this microdomain Ca²⁺ could play a role in the inhibition of adenylyl cyclase VI (AC-VI) or stimulation of Ca²⁺-dependent phosphodi-esterase (PDE), thereby lowering [cAMP].

Addendum to: