Biochemical characterization of bona fide polycystin-1 in vitro and in vivo.
The most common form of autosomal dominant polycystic kidney disease (PKD) results from mutation of the PKD1 gene on chromosome 16p13.3. The gene encodes a 14-kb messenger RNA that is predicted to express a 462-kd membrane protein. The gene product, polycystin-1, has a large extracellular portion composed of a novel combination of protein-protein interacting domains and is postulated to be a plasma membrane receptor involved in cell-cell/matrix interactions. However, slow progress has been made in the characterization of polycystin-1 or the determination of its function. In fact, the protein is expressed at very low levels in tissues and cell lines and previous efforts directed at expression of recombinant protein had been largely unsuccessful. We have recently developed constructs of full-length human PKD1 complementary (cDNA) that can be expressed in both a stable and transient fashion in mammalian cells. We used these systems to characterize our antibodies and to track the protein in vivo. We report here the first biochemical characterization of recombinant polycystin-1 and show that the protein is a 520-kd glycosylated polypeptide with an unglycosylated core of 460 kd. Subcellular fractionation as well as biotinylation studies confirmed that the protein is plasma-membrane associated. Furthermore, we show that the recombinant protein localizes to cell-cell junctions in polarized madin darby canine kidney cells as revealed by indirect immunofluorescence. Our data represent the first characterization of polycystin-1 performed under highly controlled conditions.
Boletta
A
A
Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Qian
F
F
Onuchic
L F
LF
Bragonzi
A
A
Cortese
M
M
Deen
P M
PM
Courtoy
P J
PJ
Soria
M R
MR
Devuyst
O
O
Monaco
L
L
Germino
G G
GG
D000818:Animals; D015153:Blotting, Western / Q000592:standards; D002462:Cell Membrane / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D004285:Dogs; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007365:Intercellular Junctions / Q000737:chemistry; D007668:Kidney / Q000737:chemistry; D008970:Molecular Weight; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification; D050396:TRPP Cation Channels
D018076:DNA, Complementary; D011506:Proteins; D012333:RNA, Messenger; D050396:TRPP Cation Channels