D. Bicc1 gene that is defective in the mouse model used in these studies regulates cargo-specific protein sorting mediated by the epithelial cell specific clathrin adaptor AP-1B. INTRODUCTION The nephron is the basic structural and functional unit of the kidney (Kriz and Kaissing, 2008 ). Each nephron has an initial filtering component composed of a glomerulus and Bowman’s capsule connected to a long convoluted tubule lined by transporting epithelia. Epithelial cell polarity is vitally Divalproex sodium important for correct function of different tubule segments (Kriz and Kaissing, 2008 ). In addition Divalproex sodium to the apico-basolateral axis, most renal epithelial cells exhibit planar polarity featuring primary cilia extending from the apical membrane (Fischer and Pontoglio, 2009 ). Cell polarity defects have been linked to a number of hereditary kidney diseases including polycystic kidney diseases (PKDs) characterized by the accumulation of fluid-filled cysts in the cortex and medulla (Igarashi and Somlo, 2002 ; Grantham, 2003 ; Harris and Torres, 2009 ). Approximately 50% of afflicted individuals develop end-stage renal disease requiring dialysis or kidney transplantation before the age of 60. Although presently incurable, improved understanding of disease mechanisms is uncovering new prospects for effective pathophysiology-based therapies (Torres and Harris, 2006 ). Cystic cells and tissues are also unique cell biological models for studying polarized sorting mechanisms. Human PKD susceptibility genes encode large membrane proteins. PKD1 and PKD2 are involved in the autosomal dominant form of the disease EGR1 (ADPKD), and fibrocystin is defective in autosomal recessive PKD (ARPKD; International Consortium for Polycystic Kidney Disease, 1995 ; The American Consortium for PKD1, 1995 ; Mochizuki (Amersham-Pharmacia, Piscataway, NJ). Nucleic acids coding for the dileucine motif at positions 658 and 659 were mutagenized to a di-alanine and threonine residue 654 to alanine or aspartic acid, using the EGFR-Jx/pGEX-3X template, a Quick Change Mutagenesis kit (Invitrogen, Carlsbad, CA), and the following primers (mutations in bold): 658-AA: forward, 5-AGAACACTGCGGAGGGCGGCGCAGGAGAGGGAGCTT-3 and reverse, 5-AAGCTCCCTCTCCTGCGCCGCCCTCCGCAGTGTTCT-3; T654A: forward, 5-CGTGGGATCCAAAAGAGCACTGCGGAGGCTGCTGCAG-3 and reverse, 5-CTGCAGCAGCCTCCGCAGTGCTCTCTTTTGGATCCCACG-3; and T654D: forward, 5-CGTGGGATCCAAAAGAGATCTGCGGAGGCTGCTGCAG-3 and reverse, 5-CTGCAGCAGCCTCCGCAGATCTCTCTTTTGGATCCCACG-3. The 658-AA and T654D substitutions were also introduced to full-length human EGFR cloned in the eukaryotic expression plasmid pBKlacP-CMV (Kil for 30 min to collect a crude cytosol fraction. Membrane pellets were resuspended in the NP-40 lysis buffer and incubated with 0.5 M Na2CO3 for 5 min on ice to release peripheral membrane proteins. Beads were incubated with peripheral membrane proteins for 1 h at room temperature followed by extensive washing, and bound proteins were eluted with Laemmli sample buffer and resolved by SDS-PAGE. Open in a separate window Figure 4. EGFR 658-LL basolateral sorting signal interacts with AP-1B. (A) Human-specific EGFR expression was evaluated by domain-specific EGFR1 staining of nonpermeabilized cells with well-formed tight junctions (TJ). (B) Horizontal x-z optical sections from LLCPK1 cells expressing wild-type human EGFR after domain-specific EGFR1 staining. (C) EGFR protein schematic highlights extracellular region with four subdomains (ICIV) and multiple N-glycosylation sites (?), transmembrane (TM) domain, and cytoplasmic region with juxtamembrane (Jx), kinase catalytic, and carboxyl terminal (C-term) domains. Amino acid sequences of wild-type (peptide 1 and peptide 2) and mutant (T654A, T654D, and 658-AA) peptides used in pulldown assays are shown beneath the schematic. Critical residues involved in basolateral sorting are highlighted in bold (Hobert gene defect was only recently identified (Cogswell gene products in conditionally immortalized cell lines from cystic animals and normal age-matched controls. Endogenous protein expression was analyzed Divalproex sodium with a newly developed antibody to the Bicc1 amino terminus. The gene has 22 exons encoding two splice variants in mouse kidney (Figure 1A). Exon 21 is spliced out of transcript A and transcript B encompasses the entire open reading frame (Cogswell ?/? homozygous animal (Sweeney gene has 22 exons encoding two alternatively spliced transcripts (A and B) in mouse kidney. Predicted structures for normal and disease-specific protein products shown beneath each transcript highlight RNA-binding KH (K homology) domains, exon 21 and exon 22-encoded sequences (black and gray boxes, respectively), disease-specific carboxyl-terminal extension (hatched box), and predicted molecular weights (in parenthesis). Adapted from Cogswell (2003) . (B) Equal Divalproex sodium protein aliquots were immunoblotted with Bicc1 antibody. (C) Predicted fibrocystin domain structure highlighting extracellular immunoglobulin-like TIG repeats (light gray box), transmembrane domain (hatched box), FCY-Exo and FCY-Cyt peptide antibody sequences (black box), and approximate cleavage site generating major endogenous protein species (dashed arrow). (D) Equal protein aliquots were immunoblotted with fibrocystin antibodies (FCY-Ext and FCY-Cyt described in text). (E) Cells.