Genetic disorders – Development
Kidney International (2004) 66, 1345–1355; doi:10.1111/j.1523-1755.2004.00844.x
Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm
LUÍS F C MENEZES, YIQIANG CAI, YASUYUKI NAGASAWA, ANA M G SILVA, MARY L WATKINS, ALINE M DA SILVA, STEFAN SOMLO, LISA M GUAY-WOODFORD, GREGORY G GERMINO and LUIZ F ONUCHIC
Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil; Departments of Internal Medicine and Genetics, Yale University School of Medicine, New Haven, Connecticut; Departments of Medicine and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland; Departments of Medicine and Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama; and Department of Biochemistry, University of São Paulo, São Paulo, Brazil
Correspondence: Luiz F. Onuchic, University of São Paulo School of Medicine, Division of Nephrology, Avenida Dr. Arnaldo, 455 – Sala 3310, São Paulo-SP – 01246–903 – Brazil. E-mail: lonuchic@lim12.fm.usp.br
Received 3 December 2003; Revised 16 February 2004; Accepted 7 April 2004.
Abstract
Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm.
Background
PKHD1, the autosomal-recessive polycystic kidney disease (ARPKD) gene, encodes multiple alternatively spliced transcripts predicted to generate membrane-bound and secreted proteins. The longest open reading frame encodes polyductin (fibrocystin), a putative 4074 amino acid protein with a single transmembrane domain and an intracellular C-terminus.
Methods
To characterize the PKHD1 products and their expression profile, we raised polyclonal antibodies against different portions of polyductin and analyzed different organs using various methods.
Results
Western blot analyses demonstrated specific bands of >440 kD in human adult kidney, liver, and pancreas and 
230 kD in kidney and liver, predominantly observed in membrane fractions. The >440-kD putative membrane protein was immunoprecipitated from kidney and subsequently detected by Western blotting using two distinct antisera. An additional product of 140 kD was specifically recognized by affinity-purified antisera predominantly in soluble fractions. Immunohistochemistry studies revealed specific staining in cortical and medullary collecting ducts and thick ascending limbs of Henle (TALH). Serial sections were stained with antibodies against aquaporin-2 and Tamm-Horsfall protein to confirm the nephron segment localization. Positive staining was also detected in biliary and pancreatic duct epithelia. Analyses of mouse developing tissues showed specific staining in the ureteric bud branches, intra- and extrahepatic biliary ducts, pancreatic ducts, and salivary glands. Immunofluorescence studies in inner medullary collecting duct cultured cells and immunoelectron microscopy analysis of medullary collecting ducts demonstrated that the protein localizes to the primary cilium. Positive signal was also detected in the apical membrane and in cytoplasm.
Conclusion
The results indicate that polyductin is part of the group of polycystic kidney disease (PKD)-related proteins expressed in primary apical cilia. Our data also suggest that, in addition to its likely involvement in cilia function, polyductin probably serves in other subcellular functional roles. The detection of three different products using two antisera, with evidence for distinct subcellular localizations, suggests that PKHD1 encodes membrane-bound and soluble isoforms.
Keywords:
ARPKD, PKHD1 gene, polyductin, fibrocystin, cilium, isoforms, protein expression profile
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