ALTERNATIVE MESSENGER-RNA SPLICING OF THE NOVEL GTPASE RAB28 GENERATES ISOFORMS WITH DIFFERENT C-TERMINI

A novel ras-related gene (rab28) was identified by a PCR-based cloning approach and subsequent screening of rat fat cell and brain cDNA libr aries. The deduced amino acid sequence of the cDNA is distantly relate d with members of the Rab family (31-33% sequence identity, mainly res tricted to the six GTP-binding motifs). Cloning of the human homologue of Rab28 by a PCR-based approach revealed the existence of two isofor ms (hRab28S, hRab28L) which differ only by a 95-bp insertion within th e coding region. This insertion generates an alternative sequence of t he 30 C-terminal amino acids of the protein. Both C-termini of the hum an homologues comprise farnesylation motifs, but differ strikingly in a stretch of 13 amino acids. By PCR, mRNA of hRab28S was detected in m ost tissues investigated (cortex, liver, kidney, skeletal muscle, adip ose tissue, testis and urothelium), whereas hRab28L was predominant in testis. Recombinant Rab28 proteins showed specific binding of radiola beled guanosine 5'-O-[gamma-thio]triphosphate and rapidly hydrolysed [ alpha-P-32]GTP; there was no difference in the GTP binding characteris tics of the two isoforms hRab28S and hRab28L. It is suggested that the isoforms are derived from the same gene by alternative mRNA splicing, and that their functions differ in a parameter unrelated to its basic role as a GTPase.