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

Abstract

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.

Figure 1

Mapping the heterodimerization domains of PKD1 and PKD2 (A) and the homodimerization domains of PKD2 (B) in the yeast two-hybrid system. (A) The C-terminal 226 amino acids of PKD1 and progressive C- and N-terminal deletions of this domain were inserted into pLEX. For PKD2 binding assays, the pLex bait constructs and PKD2 prey constructs were sequentially transfected into the yeast strain EGY48 bearing a lacZ reporter. Interaction (+) was indicated by β-galactosidase production and leucine prototrophy in yeast; the minimal interacting domain, PKD1.4, caused leucine prototrophy without β-galactosidase production (∗). A putative coiled–coil structure is depicted as a shaded box, a potential PEST sequence is depicted as a solid box. (B) The C-terminal 289 amino acids of PKD2 and progressive C- and N-terminal deletions of this domain were inserted into pLEX to map the interaction with PKD1 and PKD2. PKD2 binds PKD1 through the C-terminal 97 amino acids. The homodimerization of PKD2 is mediated by a region spanning amino acids 63–192, which contains a putative coiled–coil structure (shaded box).

Figure 2

Construction (A) and surface expression (B) of membrane-bound PKD1 and PKD2 fusion proteins. (A) The C-terminal 113 amino acids of PKD1 (PKD1.2) and the extracellular domain of CD16 were fused to the transmembrane region of CD7 to yield the chimeric integral membrane protein CD16.7–PKD1.2. The construct sF.3TM–PKD1, containing the last three putative transmembrane domains plus the cytoplasmic tail of PKD1, is tagged at its N terminus with the leader sequence of preprotrypsin followed by a FLAG epitope. The membrane-bound fusion of PKD2, sIg.7–PKD2, was generated by fusing the C-terminal 289 amino acids of PKD2 to a cell surface expressed immunoglobulin consisting of the leader sequence of CD5, the CH₂ and CH₃ domain of human IgG1, and the transmembrane region of CD7. (B) Surface expression of CD16.7–PKD1.2 and sIg.7–PKD2 fusion proteins. 293T cells were transfected with vector (a), CD16.7 (b), CD16.7–PKD1.2 (c), vector (d), sIg.7 (e), sIg.7–PKD2 (f), and labeled with anti-CD16-fluorescein isothiocyanate (a–c) or anti-human IgG-phycoerythrin (d–f). Photographs were taken at ×400 magnification under fluorescence microscopy 24 hr after transfection. (Insets) Lower magnification (×100) are located in the right lower of b, c, e, and f. Expression of CD16.7–PKD1.2 (c) and sIg.7–PKD2 (f) was clearly detected on the surface of unfixed cells.

Figure 3

Up-regulation of PKD1 fusion proteins by membrane-bound PKD2 (sIg.7–PKD2). 293T cells were cotransfected with 4 μg of expression vector for FLAG-tagged PKD1 or PKD2 truncations and 4 μg of expression vector for sIg.7 (control) or sIg.7–PKD2. All transfection mixes contained 2 μg of GFP. Cell lysates were immunoprecipitated with protein A, followed by sequential Western blot analysis with anti-FLAG, anti-GFP, and anti-α-catenin. Increased levels of PKD1 proteins, but not PKD2 protein, were observed (lanes 4, 6, and 8). Control for transfection efficiency was provided by GFP levels that were similar or lower than in control transfections containing sIg.7, and total amount of protein loaded in each lane was monitored by α-catenin expression.

Figure 5

Hetero- and homodimerization of PKD1 and PKD2. (A) Coimmunoprecipitations of ³⁵S-labeled PKD1 and PKD2 fusions proteins. 293T cells were transfected with sIg.7–PKD2 and F–PKD1.2 (lane 1), sIg.7–PKD2 and F–PKD2 (lane 2), sIg.7 and F–PKD1.2 (lane 3), sIg.7 and F–PKD2 (lane 4), or remained untransfected (lane 5). Cells were harvested after a 6-hr chase with [³⁵S]methionine/cysteine. Immunoprecipitations were performed with anti-FLAG M2 affinity gel. The arrow marks a putative 50-kDa protein present in lane 1 (and potentially in lanes 2 and 4), which appears to associate with PKD2 or the PKD1–PKD2 complex. Similar results were obtained with reciprocal immunoprecipitation using protein A to immunoprecipiate the IgG-tagged constructs (data not shown). (B) Coimmunoprecipitation of ³⁵S-labeled F–PKD2 with MBP–PKD1.2 fusion protein. F–PKD2 was in vitro transcribed and translated (lane 1). An aliquot of the reaction mixture was then incubated with a control MBP–protein (lane 2) or MBP–PKD1.2 (lane 3). (C) Homodimerization of PKD2 in vivo. 293T cells were cotransfected with sIg.7 and F–PKD2 (lane 1), sIg.7–CD40 and F–PKD2 (lane 2), and sIg.7–PKD2 and F–PKD2 (lane 3). Lysates were immunoprecipitated with protein A, and bound proteins were blotted with anti-FLAG. (D) Cell lysates demonstrating comparable expression of the F–PKD2 fusion protein in all three conditions (lanes 1–3 as in C).

Figure 4

Heterodimerization of PKD1 and PKD2 in vivo. (A) Coimmunoprecipitation of membrane-bound chimeric PKD1 and PKD2 proteins with cytoplasmic PKD2 and PKD1 fusion proteins, respectively. 293T cells were transfected with the indicated combinations of expression vectors for PKD1 and PKD2, and cell lysates were immunoprecipitated with protein A, followed by Western blot analysis with anti-FLAG. sIg.7–PKD1.2 interacted with F–PKD2 (lane 2) but not with the sIg.7 control protein (lane 1). The interaction between sIg.7–PKD2 and F–PKD1.2 is demonstrated in lane 4; the interaction between sIg.7–PKD2 and F–PKD1 is shown in lane 6. Neither F–PKD1.2 nor F–PKD1 interacted with the sIg.7 control protein. (B) Coimmunoprecipitation of membrane-bound fusion proteins of PKD2 and PKD1. 293T cells were transfected with the expression vectors indicated above. Immunoprecipitations were performed with protein A, followed by Western blot analysis with anti-FLAG (lanes 1–3) or anti-CD16 (lanes 4–6). Lane 1 demonstrates the interaction with sIg.7–PKD2 and sF.3TM–PKD1. No interaction was detectable between sF.3TM–PKD1 and sIg.7–CD40 or between sIg.7–PKD2 and F–BAP2. Lane 6 shows the interaction between the two membrane-bound fusion proteins CD16.7–PKD1.2 and sIg.7–PKD2. No interaction was detectable with the control proteins CD16.7 (lane 4), or sIg.7–CD40 (lane 5).