Identification of three isoforms for the Na+-dependent phosphate cotransporter (NaPi-2) in rat kidney.
We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2alpha, NaPi-2beta, and NaPi-2gamma) from a rat kidney library. NaPi-2alpha cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2beta encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2gamma encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 alpha, beta, and gamma were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2alpha and NaPi-2gamma were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2gamma and -2alpha markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter.
Tatsumi
S
S
Department of Clinical Nutrition, School of Medicine, Tokushima University, Tokushima 770, Japan.
Miyamoto
K
K
Kouda
T
T
Motonaga
K
K
Katai
K
K
Ohkido
I
I
Morita
K
K
Segawa
H
H
Tani
Y
Y
Yamamoto
H
H
Taketani
Y
Y
Takeda
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D012326:RNA Splicing; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D002352:Carrier Proteins; D018076:DNA, Complementary; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters