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. 2009 Nov;297(5):F1310-5.
doi: 10.1152/ajprenal.00412.2009. Epub 2009 Sep 2.

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2

Jozefina Casuscelli  1 , Stefan Schmidt, Brenda DeGray, Edward T Petri, Andjelka Celić, Ewa Folta-Stogniew, Barbara E Ehrlich, Titus J Boggon
Affiliations

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2

Jozefina Casuscelli et al. Am J Physiol Renal Physiol. 2009 Nov.

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, polycystin-1 (PC1) and polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca2+ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2.

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Figures

Fig. 1.
Fig. 1.
Polycystin-1 (PC1) and PC2 sequence and domain structure. A: schematic representation of PC1 and PC2 domain structure and topology. PC1 is predicted to have a >3,000-amino acid extracellular region and 11 transmembrane spans. PC2 is predicted to have 6 transmembrane spans and intracellular N- and C-termini. PC1 and PC2 are predicted to interact via their C-terminal tails. B: sequences of the PC1 and PC2 C-terminal tails showing constructs, regions of predicted secondary structure (SS-Pred) (26) and predicted coiled-coil domains (CC-Pred). Human Q4224 and mouse Q4215 are shaded in the PC1 alignment. C: schematic illustrating constructs used in this study. The locations of EF-hand and coiled-coil domains are indicated (schematics not to scale). Numbering for human PC2 and mouse PC1 are shown.
Fig. 2.
Fig. 2.
Pull downs of PC1 and PC2 C-terminal tails. A: PC2-C, PC2-EF, and PC2-CC pull downs with PC1-C. Constructs that include the coiled-coil domain of PC2 pull down with PC1 (lanes 2–7), while constructs encoding only the EF-hand domain do not (lanes 9 and 10, 12–15). Example pull downs are shown for PC2 constructs with glutathione S-transferase (GST)-PC1-4183-4270 on beads. M, marker; PC1, bound PC1 on beads; L, loaded PC2; PD, pull down on beads following 3× wash; GST, GST marker; n = ≥3. Lane numbers are used in the text. B: predicted coiled-coil domain of PC1 is sufficient to pull down PC2-C. Example pull downs are shown for 93 μM PC2-704-968 with GST-PC1-constructs on beads. M, marker; PC2 PC2 sample; B, bound PC1; L, loaded PC2; PD, pull down on beads following 3× wash; GST, GST marker; n = ≥3. Black lines indicate the intervening lanes have been spliced out. Lane numbers are used in the text.
Fig. 3.
Fig. 3.
Affinity of the cytoplasmic interaction of PC1 and PC2 C-terminal fragments. The interaction of PC2-798-927 and GST-PC1-4183-4270 produced a concentration-dependent and PC1-specific surface plasmon resonance (SPR) response [the traces presented (top) are corrected for responses generated by PC2 interacting with the chip surface that contained GST alone]. Concentrations of PC2 tested over the immobilized PC1 were 0.625, 1.25, 2.5, 5.0, and 10 μM, and the regions of the sensograms used for the steady-state analysis are marked by a horizontal bar; the errors shown (bottom) represent an average noise of ± 2 RU observed in the sensograms during data collection. The steady-state analysis (bottom) yields a K0.5 of 2.9 ± 0.9 μM. Data points are averages from duplicates, with results from 1 experiment shown (n = 3).
Fig. 4.
Fig. 4.
Polycystin-1 Q4215P mutant does not bind PC2. A: pull downs show that introduction of the Q4215P mutation into PC1-4183-4270 interrupts association with PC2-798-927. Arrow indicates PC2-798-927. PC2, PC2-704-968 loaded; B, bound PC1 on beads; PD, pull down on beads following 3× wash; GST, GST marker; n = ≥3. B: GST-PC1-C pulls down full-length PC2 from Madin-Darby canine kidney epithelial cell (MDCK) microsomes; however, introduction of the disease-related mutation Q4215P into PC1 abrogates this pull down. PC1, PC1-4183-4270; “cells,” loaded MDCK microsomes; PD, pull down on beads; Sup, supernatant following pull down. Black lines indicate the intervening lanes have been spliced out. PC2 in the supernatant was in excess of PC1 on the beads to maximize any possible interaction. Blot is representative of >3 experiments.
Fig. 5.
Fig. 5.
Expression of PC1-C, but not PC1-C-Q4215P, alters intracellular Ca2+ signaling. MDCK cells were loaded with fura 2-AM, and changes in intracellular Ca2+ levels were monitored upon addition of extracellular ATP in the absence of extracellular Ca2+. In all cases, the values plotted represent the average of the response measured in 92-254 cells from experiments performed on at least 5 different days. Control, experiments where cells were mock transfected with empty vector. A: resting Ca2+ levels were unchanged by the expression of PC1-4183-4270 (wild-type) or PC1-4183-4270-Q4215P (Q-P mutant). B: peak Ca2+ release was attenuated after addition of 3 μM ATP in cells expressing PC1-C wild-type, but not the Q-P mutant, presumably because expression of PC1-C disrupts the normal interaction between full-length PC1 and PC2 in MDCK cells. C: duration of the response to 3 μM ATP was similar in cells expressing PC1-C wild-type and the Q-P mutant. *Statistically significant (P <0.05).
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