Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1~(IP4BP) and GAP1~m result from the GAP1~(IP4BP) PH domain directing plasma membrane targeting

摘要

Inositol 1,3,4,5-tetrakisphosphate (IP_4) is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP_4 receptor, termed GAP1~(IP4BP) [1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1~(IP4BP), called GAP1~m has been identified [2] and here we describe the cloning of a GAP1~m cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1~m, in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP_4 with a similar affinity and specificity to that of the corresponding GAP1~(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1~(IP4BP) is located solely at the plasma membrane, GAP1~m seems to have a distinct perinudear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP_4.