IDENTIFICATION OF 3 ISOFORMS FOR THE NA-DEPENDENT PHOSPHATE COTRANSPORTER (NAPI-2) IN RAT-KIDNEY()

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2 alp ha, NaPi-2 beta, and NaPi-2 gamma) from a rat kidney library. NaPi-2 a lpha cDNA encodes 337 amino acids which have high homology to the N-te rminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2 beta encodes 327 amino acids which are identical to the N-terminal region o f NaPi-2 containing 4 transmembrane domains, whereas the 146 amino aci ds in the C-terminal region are completely different. In contrast, NaP i-2 gamma encodes 268 amino acids which are identical to the C-termina l half of NaPi-2, An analysis of phage and cosmid clones indicated tha t 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 polype ptides, respectively. NaPi-2 alpha: and NaPi-2 gamma were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted wi th 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa p rotein. The sizes of these proteins corresponded to those in glycosyla ted forms. A functional analysis demonstrated that NaPi-2 gamma and -2 alpha markedly inhibited NaPi-2 activity in Xenopus oocytes, The resu lts suggest that these short isoforms may function as a dominant negat ive inhibitor of the full-length transporter.