BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 143–149 (1998) ARTICLE NO. RC989179 Structural and Functional Analysis of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptors GAP1 IP4BP and GAP1 m Joanna R. Bottomley, 1 Jon S. Reynolds, 1 Peter J. Lockyer, and Peter J. Cullen 2 Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom Received June 8, 1998 isomerically specific Ins(1,3,4,5)P 4 -binding protein, which Previously we have purified and cloned a high affin- is a GAP (a GTPase-activating protein), specifically a ity isomerically specific inositol 1,3,4,5-tetrakisphos- member of the GAP1 family, and this has led us to call phate (Ins(1,3,4,5)P 4 )-binding protein which, because it GAP1 IP4BP . it is clearly a member of the GAP1 family of Ras The GAP1 family can be broadly divided into two GTPase-activating proteins (GAP), we have termed sub-families: those identical to human blood GAP1 IP4BP , GAP1 IP4BP . Here we show that expressed full-length namely bovine brain R-Ras GAP [7] and rat brain GAP- GAP1 IP4BP binds Ins(1,3,4,5)P 4 with an affinity and III [8], and homologues that are approximately 60% specificity similar to that of the originally purified pro- identical which include rat brain GAP1 m [9] and human tein, a binding activity which is dependent on a func- brain GAP1 m [10] (see [2] for a more detailed discussion tional PH/Btk domain. Furthermore, we highlight a of this family). Recently we [11] and Kobayashi et al fundamental distinction between GAP1 IP4BP and its ho- [12] have cloned the human peripheral GAP1 m from mologue GAP1 m , namely that both proteins function blood and ectocervical endothelial cells respectively. as Ras GAPs but only GAP1 IP4BP displays Rap GAP ac- tivity.  1998 Academic Press Both GAP1 IP4BP and GAP1 m appear to be ubiquitiously Key Words: Ras; Rap; GAPs; Ins(1,3,4,5)P 4 ; GAP1 IP4BP ; expressed within tissues, and generally cell lines con- GAP1 m . tain both proteins (Reynolds et al., unpublished). How- ever, we have recently established that they have fun- damentally distinct subcellular localisations, in that GAP1 IP4BP resides entirely at the plasma membrane In agonist stimulated cells inositol 1,3,4,5-tetrakisphos- [13,11], whereas GAP1 m has a perinuclear localisation phate (Ins(1,3,4,5)P 4 ) is produced directly by phosphory- with possibly a small cytosolic component [11]. lation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) via an Structurally both GAP1 IP4BP and GAP1 m possess a cen- Ins(1,4,5)P 3 3-kinase [1]. The kinetics of Ins(1,3,4,5)P 4 tral Ras GAP domain that contains a highly conserved production and degradation provided the initial evidence Ras-binding motif [14]; this is flanked by N-terminal C2A that this ubiquitious inositol phosphate may function as and C2B domains which by analogy with synaptotagmins a second-messenger. The exact second-messenger role of may constitute Ca 2/ -dependent and Ca 2/ -independent Ins(1,3,4,5)P 4 remains a matter of some controversy, both phospholipid binding domains [15], and a C-terminal as to whether it has a role at all, and if so, what that pleckstrin homology domain (PH) which is coupled to a role might be (see [2] for a review of this literature). In Bruton’s tyrosine kinase (Btk) motif [16]. In vitro both recent years we have taken the view that if Ins(1,3,4,5)P 4 GAP1 IP4BP and GAP1 m function as GAPs on N-, H-, R- does indeed constitute a novel second messenger then and K-Ras [6-10, 17,18], but interestingly when these one criteria that must be fulfilled is the presence within assays are performed in the presence of membrane phos- cells of a protein(s) which specifically binds Ins(1,3,4,5)P 4 pholipids, the GAP activity is completely dependent on i.e. an Ins(1,3,4,5)P 4 receptor [3]. To this end we have the presence of Ins(1,3,4,5)P 4 [6,18]. These data suggest described the purification [4,5] and cloning [6] of a highly therefore, that in vivo GAP1 IP4BP and GAP1 m may func- tion as Ins(1,3,4,5)P 4 -dependent Ras GAPs. 1 These two authors contributed equally to this work. Two issues regarding GAP1 IP4BP are unclear, and it is 2 To whom correspondence should be addressed at the Department these we have addressed here. Firstly, GAP1 IP4BP puri- of Biochemistry, School of Medical Sciences, University of Bristol, fied from pig platelets, but not that isolated from bovine Bristol BS8 1TD, UK. Fax: 0117 9288274. E-mail: Pete.Cullen@ bris.ac.uk. brain [7], will function not only as a Ras GAP but also 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 143