GAP1IP4BP is a member of the GAP1 family of Ras GTPase-activating proteins (

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca 2 þ ] i ) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca 2 þ ] i often occur as repetitive Ca 2 þ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca 2 þ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca 2 þ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca 2 þ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca 2 þ signals and the regulation of Ras.

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Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1 m and GAP1 IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1 m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), whereas the domain from GAP1 IP4BP binds PtdIns(3,4,5)P 3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1 m is cytosolic and undergoes a rapid PtdIns(3,4,5)P 3 -dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P 2 -dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1 IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P 2 . This has allowed the isolation of a mutant, GAP1 IP4BP -(K591T), which while retaining high affinity for PtdIns(3,4,5)P 3 has a 6-fold reduction in its affinity for PtdIns(4,5)P 2 . Importantly, GAP1 IP4BP -(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P 3 -dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1 IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1 IP4BP .Pleckstrin homology (PH) 1 domains are small ␤-sandwich protein modules of ϳ120 residues that occur once or, more rarely, several times in a protein sequence (1-4). Of those PH domains examined nearly all bind phosphoinositides present in cell membranes although with varying degrees of specificity and affinity. In the vast majority of PH domains, phosphoinositide binding is weak and quite nonspecific (1-4). However in around 10% of cases the phosphoinositide binding is strong and highly specific. For example, the PH domain of phospholipase C-␦ 1 (PLC-␦ 1 ) specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) (5, 6). In this case, the PH domain interaction with PtdIns(4,5)P 2 results in the targeting of PLC-␦ 1 to the plasma membrane (7). In contrast the PH domain from Brutons tyrosine kinase (Btk) specifically binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 (8, 9), an interaction that results in the dynamic, plasma membrane association of cytosolic Btk following the receptor-mediated production of PtdIns(3,4,5)P 3 (10).Several PH domain structures have now been solved (11-34). The core of each domain is a ␤...

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