Our Science – Randazzo Website
Paul A. Randazzo, M.D., Ph.D.
ADP-Ribosylation Factor-Directed GTPase-Activating Proteins
Coordinated remodeling of cellular membranes and the actin cytoskeleton is critical for cellular functions including protein secretion, cell migration, phagocytosis and signal transduction. Although the individual molecular machineries controlling membrane traffic and actin polymerization have been elucidated, the mechanisms coordinating the regulation of membranes and actin are largely undefined. ADP-ribosylation factor (Arf) family GTP-binding proteins and their regulators, the Arf GTPase-activating proteins (Arf GAPs) are possible links.
The Arf proteins were first identified as a cofactor for cholera toxin catalyzed ADP-ribosylation of the heterotrimeric G protein Gs. Later a family of proteins, comprised of the Arf and Arf-like (Arl) proteins was identified. The Arf proteins are divided into three classes, class I (Arf1, Arf2 and Arf3), class II (Arf4 and Arf5) and class III (Arf6). These proteins have been shown to regulate membrane traffic and to affect the actin cytoskeleton. Function of the Arf proteins is dependent on controlled binding and hydrolysis of GTP.
The Arf GTPase-activating proteins induce hydrolysis of GTP that is bound to Arf proteins. To date, fourteen mammalian Arf GAPs have been identified. A zinc finger motif comprises the catalytic domain. The Arf GAPs can be grouped into three subfamilies, Arf GAP1, the GITs and the ASAPs (see figure in gallery). The ASAPs, the focus of our laboratory, have been found to impact both actin and membrane traffic.
The ASAPs are defined by a core of a PH domain, an Arf GAP domain and ANK repeats. Four subgroups within the ASAP family are defined by domains outside of this core (see figure in gallery). ASAP1 and PAP are Arf GAPs with SH3, ANK repeat and PH domains. They also contain a proline rich domain. ACAP1/2/3 contain coiled-coil domains. AGAP1/2/3 have a GTP-binding protein like P-loop. ARAP1/2/3 have Rho GAP and multiple PH domains. Members of each subgroup have been found to affect the actin cytoskeleton and to associate with membranes. To understand the coregulation of actin and membranes, we are examining the enzymology, biochemistry and cell biology of the ASAP family Arf GAPs. Studies focus on four hypotheses about ASAP function.
Role of multiple Arf GAPs
Multiple Arfs are expressed in a single cell and a single Arf can function at multiple sites. Therefore, regulation of Arf has to be specific to both isozyme and site. We have hypothesized that these two levels of regulation are achieved in part through the action of Arf GAPs. Recent studies have identified discreet sites of action of several ASAP family Arf GAPs. To test our hypothesis, we are now examining Arf specificities of these proteins as well as structural determinants of both site and Arf specificity.
Termination of Arf signals
GTP hydrolysis on Arf is integral to its function. One aim of our work is to understand the enzymology of the Arf - Arf GAP interaction leading to GTP hydrolysis. These studies include the influence of Arf effectors. Termination of the Arf signal requires two events (i) Arf dissociate from the effector and (ii) GTP be hydrolyzed. We are examining how these two events are related and whether they are separately or integrally regulated. Our current hypothesis is that Arf - effector dissociation and Arf - GAP interaction are distinct and sequential events.
Arf GAPs as elements of signaling pathways
A third hypothesis that our laboratory is examining is that ASAP family members integrate signals from a number of pathways. The ASAP family of Arf GAPs are multidomain proteins that are targets for several signals. ASAP1, for instance, binds and is activated by signaling phospholipids (phosphatidylinositol and phosphatidic acid), binds and is phosphorylated by Src, a nonreceptor tyrosine kinase and binds the adaptor protein Crk. Current studies in the lab are aimed at understanding how the signals regulate the Arf GAPs
The role of phospholipids in Arf action is an example of how signals might be integrated. Phosphoinostide metabolism and phosphatidic acid production are affected by Arf. However, phosphoinositides and phosphatidic acid also affect Arf-target interactions and the activity of Arf regulators. As shown in the gallery, these reactions can be assembled into a system of feedforward and feedback loops that may rapidly and precipitously regulate Arf and/or phospholipid concentrations.
Other functions of Arf GAPs
A fourth hypothesis the laboratory is examining is that the ASAP family Arf GAPs may function as Arf effectors. The ASAP family Arf GAPs have complex domain structures. Some domains are involved in regulation of the GAP activity. The PH domain of ASAP1 and one of the PH domains of ARAP are clear examples. Other domains appear to have functions not related to simple inactivation of Arf. For example, PAPalpha binds to paxillin. ARAP1 has a Rho GAP domain that causes inactivation of Rho with consequent loss of stress fibers and cell rounding when ARAP1 is overexpressed. In fact, all ASAP family members that we have examined appear to affect the Rho family of GTP binding proteins. We are currently working to determine the mechanisms involved in the effects of ASAP family proteins on Rho GTP-binding proteins, whether the changes are dependent on Arf GAP activity and, for those that are not, whether the changes are dependent on Arf binding to the Arf GAP domain.
This page was last updated on 1/2/2014.