Identification of Small PDZK1-associated Protein, DD96/MAP17, as a Regulator of PDZK1 and Plasma High Density Lipoprotein Levels*

David L. Silver {ddagger} §, Nan Wang {ddagger} and Silke Vogel ¶

From the {ddagger}Department of Medicine, Division of Molecular Medicine, and the Division of Preventative Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

Received for publication, April 18, 2003 , and in revised form, May 13, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Scavenger receptor class B, type I (SR-BI) is the high density lipoprotein (HDL) receptor essential for hepatic uptake of HDL cholesterol. SR-BI was shown to impact plasma HDL levels and be anti-atherogenic. Thus, the ability to regulate hepatic SR-BI may allow for the modulation of plasma HDL cholesterol and progression of atherosclerosis. However, regulation of SR-BI in liver is not well understood. Recently, the PDZ domain containing protein PDZK1 was shown to interact with SR-BI and may serve an essential role in SR-BI cell surface expression. Here we identify an in vivo PDZK1-interacting protein that we named small PDZK1-associated protein (SPAP; also known as DD96/MAP17). Unexpectedly, we found that hepatic overexpression of SPAP in mice resulted in liver deficiency of PDZK1. The absence of PDZK1 in SPAP transgenic mice resulted in a deficiency of SR-BI in liver and markedly increased plasma HDL. Metabolic labeling experiments showed that the proteasome plays a role in the turnover of newly synthesized PDZK1, but that SPAP overexpression in liver increased PDZK1 turnover in an alternate, proteasome-independent pathway. Thus, SPAP may be an endogenous regulator of cellular PDZK1 levels by regulating PDZK1 turnover.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver is the principal site for plasma lipoprotein cholesterol uptake and elimination in bile (1). Scavenger receptor class B, type I (SR-BI)1 is a high-density lipoprotein (HDL) receptor that is highly expressed in the liver and steroidogenic tissues and at lower levels in the intestine and vasculature in adult mice (25). SR-BI is an ~82-kDa membrane glycoprotein belonging to the CD36 family of transmembrane proteins. Importantly, SR-BI plays a major role in regulating plasma HDL levels, and its activity is anti-atherogenic in mice (610). The mechanism by which SR-BI mediates hepatic lipid uptake from HDL and regulation of SR-BI activity or levels in the liver is not well understood.

Recently, the PDZ domain containing protein called PDZK1 (also known as Diphor-1 (11)/CAP70 (12)/NaPi-Cap1 (13)/CLAMP (14)) was purified from rat liver extracts by affinity chromatography using the carboxyl terminus of SR-BI (14). PDZ domains are 80–90 amino acid motifs that are often involved in scaffolding protein complexes at plasma membranes, maintaining cell polarity, and signal transduction (15). PDZK1 migrates as a 70-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, comprises four PDZ domains without other apparent protein-protein interacting or enzymatic motifs (12, 14), and is primarily expressed in kidney, liver, and lung (12). The closest homolog of PDZK1 is a protein called IKEPP (16). IKEPP is expressed in similar tissues as PDZK1 and shares a similar structure with four PDZ domains that are 30–50% identical with the PDZ domains of PDZK1 (16). Because of the presence of four PDZ domains, PDZK1 may act to scaffold a macromolecular complex at the plasma membrane. In recent studies, we have delimited the SR-BI PDZK1-interacting domain to the last three to four carboxyl-terminal amino acids of SR-BI (EAKL), defining a typical class II PDZ domain interaction (17). SR-BI deleted in the last carboxyl-terminal leucine of the PDZK1-interacting domain (SR-BIdel509) failed to interact with PDZK1 in vitro (17). Examination of transgenic mice overexpressing SR-BIdel509 in the liver revealed that SR-BIdel509 protein is not at the plasma membrane, and levels were reduced in liver, suggesting that the PDZK1-interacting domain of SR-BI is essential for expression in liver (17). On the basis of this work, we hypothesized that PDZK1 may be a key regulatory point to modulate SR-BI levels and thus plasma HDL levels, and that PDZK1-interacting proteins may be involved in regulating PDZK1 and SR-BI function. Here we describe the identification of a PDZK1 binding protein that when overexpressed in liver causes increased degradation of PDZK1, resulting in hepatic SR-BI deficiency and markedly increased plasma HDL cholesterol levels.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Polyclonal antibodies were produced to 14 amino acid carboxyl-terminal peptides of murine small PDZK1-associated protein (SPAP) and murine PDZK1 using the commercial service Multiple Peptide Systems. Anti-SR-BI polyclonal antibody was purchased from Novus Biologicals. Anti-PDZK1 monoclonal antibody used for immunoprecipitation was a kind gift from Dr. Hiroyoki Arai. IKEPP polyclonal antibody was a kind gift from Dr. Sharon Milgram. Antibodies to murine apolipoproteins E and A1 were purchased from Biodesign.

Cell Culture—HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum. SR-BI constructs were transfected into cells using LipofectAMINE 2000 (Invitrogen).

Immunoprecipitation—Liver tissue was homogenized in RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) in a Dounce homogenizer. Samples were rotated for2hat4 °C. Samples were spun at 10,000 x g for 10 min at 4 °C, then 1:100 dilutions of antibodies with protein A/G agarose beads were added to supernatants and rotated overnight at 4 °C. Samples were then washed three times with RIPA buffer, and SDS-PAGE loading buffer was added and samples were separated on SDS-PAGE for Western blot analysis.

RT-PCR—A full-length mouse SPAP cDNA was amplified from mouse liver RNA using the Access RT-PCR System (Promega) with forward primer 5'-GACAAGCTTCTGCCTGCAGCCATGTTGGCC-3' and reverse primer 5'-GACTCGAGTCACATGGGTGTGCTGCGGACC-3'. The SPAP cDNA was subcloned into the mammalian expression vector pcDNA3.1hygro+ (Invitrogen). Semi-quantitative RT-PCR was performed using the Access RT-PCR System (Promega) according to the manufacturer's protocol. Total RNA from mouse tissues was used in RT-PCR reactions to amplify full-length SPAP cDNA using the same primers shown above. Mouse {beta}-actin was amplified with forward primer 5'-AGAGGGAAATCGTGCGTGAC-3' and reverse primer 5'-CAATAGTGATGACCTGGCCGT-3'.

Generation of SPAP-expressing Adenovirus—The mouse full-length cDNA for SPAP was subcloned into the shuttle vector pCR259 (Q-Biogen), and adenovirus was then produced using the Transpose-Ad kit from Q-Biogen.

Generation of SPAP Transgenic Mice—SPAP transgenic mice were produced using the same liver-specific expression cassette as described previously (18). Briefly, a PmeI-XhoI fragment containing the murine SPAP cDNA was excised from pcDNA3.1 and subcloned into the pLIV-7 plasmid. A linearized fragment of the construct containing the promoter, first exon, first intron, and part of the second exon of the human apoE gene; the SR-BIdel509 cDNA and the polyadenylation sequence; and hepatic control region of the apoE/C-I gene locus were used to generate transgenic mice by standard procedures. Founder animals were backcrossed to C57Bl/6J mice, and two transgenic mouse lines, 261 and 263, were established. Studies in this paper were performed using mice, 8–12 weeks of age, from line 263 genotypically positive for the SPAP transgene versus control littermates negative for the SPAP transgene. All mice were fed a standard chow diet. All animal procedures were approved by the Institutional Animal Care and Research Advisory Committee at Columbia University.

Metabolic Labeling—Primary hepatocytes were isolated according to Silver et al. (19) and allowed to attach to plates for 1.5 h in DMEM 5% fetal bovine serum. Hepatocytes were then pre-incubated with methionine/cysteine-free DMEM for 1.5 h before pulse-labeling with 100 µCi/ml of [35S]methionine/cysteine for 30 min. Cells were subsequently washed and chased in DMEM 5% fetal bovine serum with 10x more methionine/cysteine for the indicated times. A concentration of lactacystin at 20 µM in Me2SO (Calbiochem), a concentration commonly used to specifically inhibit the proteasome, was added to cells after the initial pulse period for 60 min to allow cells to achieve steady state and for the duration of the chase period. Control cells received a similar concentration of Me2SO.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify PDZK1-interacting partners, immunoprecipitation experiments were carried out with anti-PDZK1 antibody after overexpressing PDZK1 using adenovirus in mouse primary hepatocytes. The rationale is that overexpression of PDZK1 may lead to increased complexes between PDZK1 and PDZK1-interacting proteins that can be co-immunoprecipitated and identified on SDS-PAGE gels. Primary mouse hepatocytes that overexpressed PDZK1 from the adenovirus were metabolically labeled with [35S]methionine, and PDZK1 immunoprecipitated from hepatocyte lysates. These experiments resulted in the weak appearance on SDS-PAGE gels of an ~14-kDa protein from PDZK1-overexpressing liver in addition to the 70-kDa PDZK1 band (Fig. 1), which we have designated SPAP. Further purification steps necessary to identify SPAP proved difficult. However, this protein was similar in size to the previously identified PDZK1-interacting protein DD96/MAP17 (2022). SPAP/DD96/MAP17 is predicted to be a type I transmembrane protein having a class I PDZ-interacting domain (STMP) at the carboxyl terminus (Fig. 2A). DD96/MAP17 was shown to be expressed in epithelial cells of kidney and breast, as well as carcinomas derived from these organs, but expression in liver has not been examined previously (20). We found SPAP/DD96/MAP17 to be expressed primarily in kidney, lung, and liver of adult mouse tissues (Fig. 1B), the same tissues that express PDZK1 (12). Although experiments using mammalian two-hybrid or binding assays on nitrocellulose membranes in vitro suggested that DD96/MAP17 interacts with PDZK1 (22), an in vivo function and physiological role for binding with PDZK1 were unknown previously.



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FIG. 1.
A 14-kDa protein co-immunoprecipitates with PDZK1 in mouse primary hepatocytes. Mouse primary hepatocytes were metabolically labeled with [35S]methionine/cysteine after a 24-h infection with an adenovirus expressing PDZK1. PDZK1 was immunoprecipitated using a polyclonal PDZK1 antibody. The blot on the right is an overexposure to better visualize the 14-kDa protein in the PDZK1 immunoprecipitate. Control immunoprecipitation was performed with pre-immune polyclonal antibody. The 14-kDa protein was reproducibly observed, compared with other bands in immunoprecipitation experiments. Arrowhead, 14-kDa protein.

 


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FIG. 2.
Mouse SPAP protein sequence and expression pattern in mouse tissues. A, protein sequence of murine SPAP (DD96/MAP17, accession number AK008253 [GenBank] ). Amino acids in bold, PDZ-interacting domain; underlined amino acids, putative transmembrane domain. B, the upper panel shows a semi-quantitative RT-PCR of SPAP expression levels in mouse tissues. Ethidium bromide gel shows full-length SPAP (upper panel). Duplicate samples of RNA were analyzed for {beta}-actin levels as a control (middle panel). Lower panel shows SPAP protein levels in 50 µg of protein extract from the indicated mouse tissues. br, brain; h, heart; k, kidney; ad, adrenal gland; li, liver; si, small intestine; lu, lung. C, SPAP co-immunoprecipitates with PDZK1 from both kidney and liver. PDZK1 monoclonal antibody or control monoclonal antibody ({beta}-actin) were used for immunoprecipitation. Immunoprecipitates were probed with polyclonal anti-PDZK1 and SPAP antibodies. IP, immunoprecipitate.

 

To provide direct evidence that endogenously expressed SPAP in mouse tissues interacts with PDZK1 in vivo, PDZK1 immunoprecipitation was performed from mouse kidney and liver. Interaction in kidney was also examined because of the high levels of expression of both PDZK1 and SPAP in this tissue. Fig. 2C shows that SPAP and PDZK1 co-immunoprecipitated from kidney and liver. More SPAP was co-immunoprecipitated from kidney, reflecting higher expression levels of PDZK1 in that tissue relative to liver (12). Taken together, the data indicate that SPAP and PDZK1 are expressed endogenously in liver and interact in vivo.

To test the in vivo effect of SPAP on PDZK1 and SR-BI in liver, liver-specific overexpression of SPAP was achieved using a human apoE/liver-specific enhancer expression cassette shown to give liver-specific transgene expression (23). We used this vector system previously to successfully produce liver-specific overexpression of SR-BI and SR-BIdel509 in mice (17, 18).

Surprisingly, SPAP overexpression resulted in the absence of PDZK1 in liver (Fig. 3A). Importantly, overexpression of SPAP did not result in decreased levels of the PDZK1 homolog IKEPP, indicating specificity toward PDZK1 (Fig. 3A). Thus, SPAP overexpression in liver resulted in hepatic deficiency of PDZK1. On the basis of our previous experiments indicating that PDZK1 is essential for SR-BI expression in liver, we hypothesized that livers of SPAP transgenic mice would have a deficiency in SR-BI. Indeed, SR-BI protein levels in livers of SPAP transgenic mice were markedly reduced (<10% of wild-type levels) but not in adrenal glands or in peritoneal macrophages (Fig. 3A), confirming our previous findings and providing evidence that PDZK1 is essential for SR-BI expression in liver (17). Both PDZK1 and SR-BI mRNA levels were unchanged in SPAP transgenic mice, indicating that SPAP regulation of PDZK1 is post-transcriptional (Fig. 3B), and that SPAP expression likely causes degradation of PDZK1. It is important to note that SR-BI gene-targeted mice have normal levels of liver PDZK1 (24), supporting our findings that PDZK1 is essential for SR-BI expression in liver, but not vice versa.



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FIG. 3.
Livers from SPAP transgenic mice (mice are littermates) are deficient in PDZK1 and SR-BI. A, Western blot analysis of liver, adrenal, and peritoneal macrophage lysates from SPAP transgenic and wild-type littermate mice. PDZK1 is absent, and SR-BI decreased in livers from SPAP transgenic mice. SR-BI levels are normal in adrenals and peritoneal macrophages from SPAP transgenic mice. B, Northern blot analysis on RNA from livers of SPAP transgenic and littermate mice indicates no change in SR-BI or PDZK1 mRNA. +, SPAP transgenic; –, non-transgenic littermate.

 

To examine whether SPAP overexpression results in increased degradation of PDZK1, we performed metabolic pulse-chase assays on primary hepatocytes isolated from SPAP transgenic mice and wild-type littermates. Fig. 4A indicates that PDZK1 turnover in hepatocytes from SPAP transgenic mice is faster than that measured in hepatocytes from wild-type littermates (relative turnover of PDZK1 in SPAP transgenic mice was greater than 3-fold faster than in wild-type littermates). Steady-state levels of PDZK1 were not detected in hepatocytes from SPAP transgenic mice (Fig. 4A, lower panel), as also observed for whole liver extracts (Fig. 3A). We did not detect a change in SR-BI turnover in isolated primary hepatocytes from SPAP transgenic mice compared with wild-type littermates (data not shown). A common pathway by which cellular proteins are degraded is via the proteasome (25). To determine whether SPAP-mediated degradation of PDZK1 requires a functional proteasome, we performed metabolic pulse-chase assays on isolated hepatocytes in the presence or absence of the specific proteasome inhibitor lactacystin. Fig. 4B shows that lactacystin blocked the turnover of PDZK1 in hepatocytes from wild-type littermates but not from SPAP transgenic mice. Lactacystin treatment was effective in blocking the proteasome-mediated degradation of PDZK1 in hepatocytes from wild-type littermates because pulse-labeled PDZK1 was found to be increased relative to non-treated hepatocytes (64% increase in PDZK1, p < 0.025; Fig. 4B). Steady-state levels of PDZK1 (Western blot, Fig. 4B) were not significantly increased after lactacystin treatment. Again, PDZK1 at steady state could not be detected in hepatocytes from SPAP transgenic mice (Western blot, Fig. 4). Together, the data indicate that turnover of PDZK1 in wild-type hepatocytes occurs in part by a proteasome-dependent pathway, whereas SPAP overexpression in liver leads to rapid degradation of PDZK1 by a proteasome-independent pathway, which in turn results in the posttranslational down-regulation of SR-BI.



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FIG. 4.
Posttranslational regulation of PDZK1. A, freshly isolated primary hepatocytes from SPAP transgenic mice and wild-type littermates were metabolically pulse-labeled and chased for the indicated times. Labeled, immunoprecipitated PDZK1 undergoing degradation during the chase period was visualized and measured by PhosphorImager analysis (upper panel). Steady-state levels of PDZK1 were determined by Western blot analysis (lower panel). B, primary hepatocytes were metabolically pulse-chased and labeled as in A, and cells were treated or not treated with the proteasome inhibitor lactacystin during the chase period (see "Experimental Procedures"). Labeled immunoprecipitated PDZK1 undergoing degradation during the chase period was visualized and measured by PhosphorImager analysis (top panel). The percentage of labeled PDZK1 remaining relative to time zero for each treatment is shown under the blot. Steady-state levels of PDZK1 were determined by Western blot analysis (lower panel). cys/met, cysteine/methionine; Lact, lactacystin.

 

SR-BI knock-out mice have a 2-fold increase in plasma HDL relative to wild-type mice (26). Although SR-BI is expressed in multiple tissues, the liver is the major site of HDL cholesterol metabolism, and therefore, we expected that the effect of a liver-specific deficiency of SR-BI found in SPAP transgenic mice would result in increased plasma HDL. Indeed, analysis of plasma cholesterol from SPAP transgenic mice revealed a 2-fold increase in cholesterol levels compared with wild-type littermates (Fig. 5A). The marked increase in plasma cholesterol determined by fast protein liquid chromatography (FPLC) analysis was attributable solely to an increase in HDL cholesterol in SPAP transgenic mice (Fig. 5B). The FPLC profile reveals that a portion of the HDL in SPAP transgenic mice is large "low density lipoprotein (LDL)-like" HDL enriched in apoE relative to apoAI (Fig. 5C, fractions 17–20). Thus, SPAP raises plasma HDL cholesterol by down-regulating PDZK1 levels in liver that, in turn, down-regulates SR-BI.



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FIG. 5.
Analysis of plasma cholesterol in SPAP transgenic mice. A, SPAP transgenic mice have increased plasma cholesterol. Values are mean ± S. D.; WT versus SPAP, p < 0.001, n = 5 for each genotype. B, FPLC analysis (31) of plasma cholesterol and triglyceride in SPAP transgenic mice versus wild-type littermates. n = 5 for each genotype. WT, wild-type littermates; SPAP, SPAP transgenic mice. C, Western blot analysis of apoE and apoA1 from fraction obtained from FPLC analysis shown in B. +, SPAP transgenic; –, non-transgenic littermate.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified an endogenous 14-kDa PDZK1-interacting protein by immunoprecipitation assays with PDZK1 in mouse liver. We named this protein SPAP. SPAP is of similar size to the previously identified PDZK1-interacting protein DD96/MAP17. However, DD96/MAP17 was only reported to interact with PDZK1 in mammalian two-hybrid or binding assays on nitrocellulose membranes in vitro but not shown to interact with PDZK1 in vivo (22). DD96 was identified as an up-regulated differential display product from a human renal carcinoma compared with normal renal parenchyma (20). On the basis of hydropathical analysis, DD96 was redesignated as membrane-associated protein 17 kDa (MAP17) (21). Thus, an in vivo function and physiological role of DD96/MAP17 binding with PDZK1 was unknown previously. In our studies reported here, we show that DD96/MAP17 is expressed in liver and co-immunoprecipitated with PDZK1 from mouse liver, indicating an in vivo interaction. Therefore, we suggest renaming DD96/MAP17 as SPAP, which better describes its function. We examined the in vivo function of SPAP in liver by overexpressing SPAP under the control of the apoE promoter and liver-specific enhancer (23). The overexpression of genes involved in HDL metabolism (e.g., SR-BI (18, 27, 28), ABCA1 (29), apoA1 (30), phospholipid transfer protein (31), hepatic lipase (32), and endothelial lipase (33)) has been a successful approach used initially to reveal in vivo functions that were later confirmed by knock-out mouse models (3438).

SPAP overexpression in mouse liver resulted in the unexpected finding that PDZK1 was undetectable in livers of SPAP transgenic mice. Importantly, SPAP overexpression did not reduce levels of the PDZK1 homolog IKEPP in liver, indicating a level of ligand specificity of SPAP for PDZK1. In metabolic pulse-chase assays to examine PDZK1 degradation, we found that PDZK1 turnover is enhanced 3-fold in hepatocytes from SPAP transgenic mice relative to non-transgenic littermates, likely explaining the PDZK1 deficiency in livers from SPAP transgenic mice. In the same pulse-chase experiments, we did not find any change in SR-BI turnover in hepatocytes from SPAP transgenic mice relative to wild-type littermates. This result was not surprising because the PDZK1-interacting domain is not essential for SR-BI expression in non-polarized cells (39, 40) but is essential for SR-BI expression in polarized hepatocytes in liver (17). Treatment of hepatocytes with lactacystin, a specific inhibitor of proteasomal degradation, inhibited turnover of PDZK1 in wild-type littermates but not in hepatocytes from SPAP transgenic mice, indicating a physiological role of the proteasome pathway in the regulation of turnover of PDZK1. However, PDZK1 steady-state levels in wild-type hepatocytes treated with lactacystin were not significantly increased relative to PDZK1 in untreated cells. In addition, steady-state levels of PDZK1 are undetectable in livers and isolated hepatocytes from SPAP transgenic mice. These data suggest that proteasome-mediated degradation of PDZK1 may account for only a small pool of cellular PDZK1, namely newly synthesized PDZK1, whereas other pathways, such as that mediated by SPAP, may play a major role in PDZK1 turnover. Turnover of a single protein by multiple degradation pathways has been reported. For example, newly synthesized cystic fibrosis transmembrane conductance regulator (CFTR) protein, a known in vivo partner of PDZK1 in kidney (12), is degraded by the proteasome, whereas CFTR that reaches the plasma membrane can undergo rapid endocytosis and sorting to the lysosome for degradation (41). The nature of the SPAP-mediated PDZK1 degradation pathway will be explored in future studies. Lastly, we showed that livers of SPAP transgenic mice are deficient in SR-BI, correlating with markedly increased plasma HDL levels in SPAP transgenic mice.

Interestingly, PDZK1 has a putative PEST sequence (identified by the program PESTfind (42); PEST score of +12.63; threshold of significance is +5.0) located between amino acids 350 and 374, which is between PDZ domains 3 and 4. PEST sequences are proline-, glutamate-, serine-, and threonine-rich sequences that are signals for proteasome-mediated degradation (43). Whether the putative PEST sequence of PDZK1 is functional and required for the proteasome-mediated or SPAP-mediated degradation will be explored in future studies.

Previously, we showed that a mutant SR-BI lacking the carboxyl-terminal PDZK1-interacting domain (SR-BIdle509) is not expressed on the cell surface of hepatocytes and is down-regulated by a post-transcriptional mechanism (17). On the basis of that study, it was not unexpected that livers of SPAP transgenic mice would be deficient in SR-BI, and plasma HDL cholesterol would be increased. This result is supported by recent data showing that mice with targeted disruption of the PDZK1 gene have similarly increased plasma cholesterol (44) as SPAP transgenic mice. However, Kocher et al. (44) did not report levels of HDL cholesterol nor present a mechanism for increased plasma cholesterol in PDZK1 knock-out mice. In addition, our findings reveal that SR-BI expression in liver but not in other tissues, such as adrenal gland, plays a major role in regulating plasma HDL levels. The FPLC profile of plasma cholesterol was similar to that seen for SR-BI knock-out mice (26), with increased HDL3 and the appearance of large LDL-like HDL. This LDL-like HDL (fractions 17–20) is enriched in apoE. This apoE-rich HDL is what Krieger and co-workers (45) called "toxic" lipoproteins, which is proposed to be responsible for the infertility in female SR-BI knock-out mice. The majority of HDL appears to be of normal size, but increased in quantity, as indicated by increased HDL apolipoprotein (apoAI and apoE) content proportional to increased cholesterol (fractions 21–28).

PDZK1 has four PDZ domains. Each may bind a specific protein at any given time, potentially forming a large macromolecular complex such as those found for other PDZ domain proteins (15). The reported PDZK1 binding partners are CFTR, which was shown to interact with PDZK1 in immunoprecipitation experiments from kidney extracts (12), type IIa Na/Pi co-transporter (13), and the multidrug resistance protein 2 (MRP2; cMOAT) (46). Thus far, only CFTR has been shown to interact in vivo with PDZK1, whereas the other aforementioned proteins have been shown to interact with PDZK1 in yeast/two-hybrid assays. MRP2 deficiency (Dubin-Johnson syndrome) is characterized primarily by hyperbilirubinemia (47). Mice with deficiency in radixin, the most abundant ERM (erzin, radixin, moesin) protein in liver, have a deficiency of MRP2 from the canalicular membrane and exhibit hyperbilirubinemia (48). On the basis of the findings that PDZK1 is not localized to canalicular membranes in liver (14) and that SPAP transgenic mice do not have hyperbilirubinemia (data not shown), despite PDZK1 deficiency, indicates that PDZK1 is not essential for MRP2 function in vivo in liver.

We hypothesize that other PDZK1 partners may be similarly down-regulated, similar to SR-BI, or be mislocalized in livers of SPAP transgenic mice. Therefore, to generate a more complete mechanistic picture of the function of SPAP and PDZK1 in liver, it will be necessary to identify other partners of PDZK1 in liver. It is tempting to speculate that signals or factors that may regulate SPAP may contribute to variation in plasma HDL cholesterol levels by modulating PDZK1 and SR-BI levels in liver. In conclusion, we have identified SPAP (DD96/MAP17) as an in vivo partner of PDZK1 that may act as a regulator of cellular PDZK1 levels.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AK008253 [GenBank] .

* This work was supported by Grant AHA0130305N (to D. L. S.) from the American Heart Association and Pfizer International HDL Research Award CU516105. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Medicine, P&S Rm. 8-401, Columbia University College of Physicians and Surgeons, 630 W. 168 St., New York, NY 10032. E-mail: dls51{at}columbia.edu.

1 The abbreviations used are: SR-BI, scavenger receptor class B, type I; HDL, high density lipoprotein; LDL, low density lipoprotein; SPAP, small PDZK1-associated protein; RT, reverse transcription; DMEM, Dulbecco's modified Eagle's medium; MAP17, membrane-associated protein 17 kDa; FPLC, fast protein liquid chromatography; CFTR, cystic fibrosis transmembrane conductance regulator; MRP2, multidrug resistance protein 2; IKEPP, intestinal and kidney-enriched PDZ protein. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Glass, C., Pittman, R. C., Weinstein, D. B., and Steinberg, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5435–5439[Abstract]
  2. Uittenbogaard, A., Shaul, P. W., Yuhanna, I. S., Blair, A., and Smart, E. J. (2000) J. Biol. Chem. 275, 11278–11283[Abstract/Free Full Text]
  3. Yuhanna, I. S., Zhu, Y., Cox, B. E., Hahner, L. D., Osborne-Lawrence, S., Lu, P., Marcel, Y. L., Anderson, R. G., Mendelsohn, M. E., Hobbs, H. H., and Shaul, P. W. (2001) Nat. Med. 7, 853–857[CrossRef][Medline] [Order article via Infotrieve]
  4. Hauser, H., Dyer, J. H., Nandy, A., Vega, M. A., Werder, M., Bieliauskaite, E., Weber, F. E., Compassi, S., Gemperli, A., Boffelli, D., Wehrli, E., Schulthess, G., and Phillips, M. C. (1998) Biochemistry 37, 17843–17850[CrossRef][Medline] [Order article via Infotrieve]
  5. Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Science 271, 518–520[Abstract]
  6. Braun, A., Trigatti, B. L., Post, M. J., Sato, K., Simons, M., Edelberg, J. M., Rosenberg, R. D., Schrenzel, M., and Krieger, M. (2002) Circ. Res. 90, 270–276[Abstract/Free Full Text]
  7. Arai, T., Wang, N., Bezouevski, M., Welch, C., and Tall, A. R. (1999) J. Biol. Chem. 274, 2366–2371[Abstract/Free Full Text]
  8. Huszar, D., Varban, M. L., Rinninger, F., Feeley, R., Arai, T., Fairchild-Huntress, V., Donovan, M. J., and Tall, A. R. (2000) Arterioscler. Thromb. Vasc. Biol. 20, 1068–1073[Abstract/Free Full Text]
  9. Kozarsky, K. F., Donahee, M. H., Glick, J. M., Krieger, M., and Rader, D. J. (2000) Arterioscler. Thromb. Vasc. Biol. 20, 721–727[Abstract/Free Full Text]
  10. Trigatti, B., Rayburn, H., Vinals, M., Braun, A., Miettinen, H., Penman, M., Hertz, M., Schrenzel, M., Amigo, L., Rigotti, A., and Krieger, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9322–9327[Abstract/Free Full Text]
  11. Custer, M., Spindler, B., Verrey, F., Murer, H., and Biber, J. (1997) Am. J. Physiol. 273, F801–F806[Medline] [Order article via Infotrieve]
  12. Wang, S., Yue, H., Derin, R. B., Guggino, W. B., and Li, M. (2000) Cell 103, 169–179[Medline] [Order article via Infotrieve]
  13. Gisler, S. M., Stagljar, I., Traebert, M., Bacic, D., Biber, J., and Murer, H. (2001) J. Biol. Chem. 276, 9206–9213[Abstract/Free Full Text]
  14. Ikemoto, M., Arai, H., Feng, D., Tanaka, K., Aoki, J., Dohmae, N., Takio, K., Adachi, H., Tsujimoto, M., and Inoue, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6538–6543[Abstract/Free Full Text]
  15. Hung, A. Y., and Sheng, M. (2002) J. Biol. Chem. 277, 5699–5702[Free Full Text]
  16. Scott, R. O., Thelin, W. R., and Milgram, S. L. (2002) J. Biol. Chem. 277, 22934–22941[Abstract/Free Full Text]
  17. Silver, D. L. (2002) J. Biol. Chem. 277, 34042–34047[Abstract/Free Full Text]
  18. Wang, N., Arai, T., Ji, Y., Rinninger, F., and Tall, A. R. (1998) J. Biol. Chem. 273, 32920–32926[Abstract/Free Full Text]
  19. Silver, D. L., Wang, N., and Tall, A. R. (2000) J. Clin. Invest. 105, 151–159[Abstract/Free Full Text]
  20. Kocher, O., Cheresh, P., Brown, L. F., and Lee, S. W. (1995) Clin. Cancer Res. 1, 1209–1215[Abstract]
  21. Kocher, O., Cheresh, P., and Lee, S. W. (1996) Am. J. Pathol. 149, 493–500[Abstract]
  22. Kocher, O., Comella, N., Tognazzi, K., and Brown, L. F. (1998) Lab. Invest. 78, 117–125[Medline] [Order article via Infotrieve]
  23. Simonet, W. S., Bucay, N., Pitas, R. E., Lauer, S. J., and Taylor, J. M. (1991) J. Biol. Chem. 266, 8651–8654[Abstract/Free Full Text]
  24. Mardones, P., Pilon, A., Bouly, M., Duran, D., Nishimoto, T., Arai, H., Kozarsky, K. F., Altayo, M., Miquel, J. F., Luc, G., Clavey, V., Staels, B., and Rigotti, A. (2003) J. Biol. Chem. 278, 7884–7890[Abstract/Free Full Text]
  25. Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998) Cell 92, 367–380[Medline] [Order article via Infotrieve]
  26. Rigotti, A., Trigatti, B. L., Penman, M., Rayburn, H., Herz, J., and Krieger, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12610–12615[Abstract/Free Full Text]
  27. Kozarsky, K. F., Donahee, M. H., Rigotti, A., Iqbal, S. N., Edelman, E. R., and Krieger, M. (1997) Nature 387, 414–417[CrossRef][Medline] [Order article via Infotrieve]
  28. Ueda, Y., Royer, L., Gong, E., Zhang, J., Cooper, P. N., Francone, O., and Rubin, E. M. (1999) J. Biol. Chem. 274, 7165–7171[Abstract/Free Full Text]
  29. Vaisman, B. L., Lambert, G., Amar, M., Joyce, C., Ito, T., Shamburek, R. D., Cain, W. J., Fruchart-Najib, J., Neufeld, E. D., Remaley, A. T., Brewer, H. B., Jr., and Santamarina-Fojo, S. (2001) J. Clin. Invest. 108, 303–309[Abstract/Free Full Text]
  30. Walsh, A., Ito, Y., and Breslow, J. L. (1989) J. Biol. Chem. 264, 6488–6494[Abstract/Free Full Text]
  31. Jiang, X., Francone, O. L., Bruce, C., Milne, R., Mar, J., Walsh, A., Breslow, J. L., and Tall, A. R. (1996) J. Clin. Invest. 98, 2373–2380[Abstract/Free Full Text]
  32. Fan, J., Wang, J., Bensadoun, A., Lauer, S. J., Dang, Q., Mahley, R. W., and Taylor, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8724–8728[Abstract]
  33. Jaye, M., Lynch, K. J., Krawiec, J., Marchadier, D., Maugeais, C., Doan, K., South, V., Amin, D., Perrone, M., and Rader, D. J. (1999) Nat. Genet. 21, 424–428[CrossRef][Medline] [Order article via Infotrieve]
  34. Plump, A. S., Azrolan, N., Odaka, H., Wu, L., Jiang, X., Tall, A., Eisenberg, S., and Breslow, J. L. (1997) J. Lipid Res. 38, 1033–1047[Abstract]
  35. Jin, W., Millar, J. S., Broedl, U., Glick, J. M., and Rader, D. J. (2003) J. Clin. Invest. 111, 357–362[Abstract/Free Full Text]
  36. Ishida, T., Choi, S., Kundu, R. K., Hirata, K., Rubin, E. M., Cooper, A. D., and Quertermous, T. (2003) J. Clin. Invest. 111, 347–355[Abstract/Free Full Text]
  37. Jiang, X. C., Bruce, C., Mar, J., Lin, M., Ji, Y., Francone, O. L., and Tall, A. R. (1999) J. Clin. Invest. 103, 907–914[Abstract/Free Full Text]
  38. Homanics, G. E., de Silva, H. V., Osada, J., Zhang, S. H., Wong, H., Borensztajn, J., and Maeda, N. (1995) J. Biol. Chem. 270, 2974–2980[Abstract/Free Full Text]
  39. Connelly, M. A., de la Llera-Moya, M., Monzo, P., Yancey, P. G., Drazul, D., Stoudt, G., Fournier, N., Klein, S. M., Rothblat, G. H., and Williams, D. L. (2001) Biochemistry 40, 5249–5259[Medline] [Order article via Infotrieve]
  40. Gu, X., Trigatti, B., Xu, S., Acton, S., Babitt, J., and Krieger, M. (1998) J. Biol. Chem. 273, 26338–26348[Abstract/Free Full Text]
  41. Gelman, M. S., and Kopito, R. R. (2002) J. Clin. Invest. 110, 1591–1597[Free Full Text]
  42. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364–368[Medline] [Order article via Infotrieve]
  43. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem. Sci. 21, 267–271[CrossRef][Medline] [Order article via Infotrieve]
  44. Kocher, O., Pal, R., Roberts, M., Cirovic, C., and Gilchrist, A. (2003) Mol. Cell. Biol. 23, 1175–1180[Abstract/Free Full Text]
  45. Miettinen, H. E., Rayburn, H., and Krieger, M. (2001) J. Clin. Invest. 108, 1717–1722[Abstract/Free Full Text]
  46. Kocher, O., Comella, N., Gilchrist, A., Pal, R., Tognazzi, K., Brown, L. F., and Knoll, J. H. (1999) Lab. Invest. 79, 1161–1170[Medline] [Order article via Infotrieve]
  47. Keppler, D., and Konig, J. (2000) Semin. Liver Dis. 20, 265–272[CrossRef][Medline] [Order article via Infotrieve]
  48. Kikuchi, S., Hata, M., Fukumoto, K., Yamane, Y., Matsui, T., Tamura, A., Yonemura, S., Yamagishi, H., Keppler, D., and Tsukita, S. (2002) Nat. Genet. 31, 320–325[CrossRef][Medline] [Order article via Infotrieve]