Characterization of PKD protein-positive exosome-like vesicles.
Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.
Hogan
Marie C
MC
Division of Nephrology & Hypertension, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Manganelli
Luca
L
Woollard
John R
JR
Masyuk
Anatoliy I
AI
Masyuk
Tatyana V
TV
Tammachote
Rachaneekorn
R
Huang
Bing Q
BQ
Leontovich
Alexey A
AA
Beito
Thomas G
TG
Madden
Benjamin J
BJ
Charlesworth
M Cristine
MC
Torres
Vicente E
VE
LaRusso
Nicholas F
NF
Harris
Peter C
PC
Ward
Christopher J
CJ
D000818:Animals; D019921:Biotinylation; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D007690:Polycystic Kidney Diseases / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D050396:TRPP Cation Channels / Q000378:metabolism
D020543:Proteome; D011956:Receptors, Cell Surface; D050396:TRPP Cation Channels; C451750:PKHD1 protein, human; C484365:Pkhd1 protein, mouse