ReviewThe PIX–GIT complex: A G protein signaling cassette in control of cell shape
Introduction
Ras-like G proteins (small G proteins) and the molecules that regulate their activity serve as potent determinants of cell shape by impacting on the cytoskeleton, membrane dynamics, and vesicular trafficking [1], [2], [3], [4]. In general, small G proteins cycle between an active, GTP-bound, and an inactive, GDP-bound form, and they require precise spatiotemporal activation in order to execute their proper function [5]. Accordingly, the use of live cell activation state-specific fluorescent probes in fluorescence resonance energy transfer (FRET) has demonstrated that the localization of GTP-bound forms of small G proteins is indeed is highly restricted within the cell [6], [7], [8]. This is accomplished by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which in addition to enzymatic activity, possess protein–protein and phosphoinositide interacting domains that restrict their function to defined subcellular locations [9], [10]. Moreover, GEFs and GAPs often mediate cross talk between members of the same or distinct families of small GTP-binding proteins. Such mechanisms permit coordination between, for instance, vesicular trafficking and cytoskeletal remodeling controlled by Arf and Rho proteins, respectively. Here, we discuss the role of the Cdc42/Rac exchange factor PIX, and the ArfGAP GIT in regulating cell shape and polarity in response to integrin ligation, G protein-coupled receptor signaling, receptor tyrosine kinase activation, and other stimuli. While PIX and GIT proteins have recently been reviewed individually [11], [12], [13], there are compelling reasons for discussing their roles in morphogenetic events collectively, as they form a tight complex in cells and influence the stability and function of one another. Indeed, it is becoming apparent that the PIX–GIT complex operates as a cassette that exerts its physiological functions by coordinating the localization and activation of Arf and Rho proteins.
Section snippets
PIX: PAK-interacting exchange factor
The PIX, a.k.a. ’Cool’, proteins were isolated as direct interactors with the Cdc42/Rac effector molecule PAK [14], [15], and were also identified in a screen for SH3 domain-containing proteins [16]. Of the two forms, αPIX is expressed primarily in hematopoetic cells and muscle [14], whereas βPIX is ubiquitously expressed and is subject to extensive splicing, particularly in the brain [14], [17]. PIX molecules are Dbl-family exchange factors for Rho proteins, defined by the presence of tandem
GIT: G protein-coupled receptor kinase interactor
GIT1 was initially identified as a direct interactor with GRK, G protein-coupled receptor kinase [33], where it regulates Arf6-dependent trafficking of β2-adrenergic receptors [33], [34]. GIT proteins exist in two forms, GIT1 and GIT2, which also have been named CATs (Cool-associated tyrosine phosphorylated protein) [20], PKL (Paxillin-kinase linker) [21], and p95-APP (ArfGAP-putative, PIX-interacting, Paxillin-interacting proteins) [23] (Fig. 2). Apparently, only one form of GIT1 exists,
Role of the PIX–GIT complex in membrane ruffling and cell spreading
The PIX–GIT complex has been most widely studied in the context of integrin-mediated cell spreading and cell motility. Focal adhesions are macromolecular protein and phosphoinositide assemblies on the inner leaflet of the plasma membrane that connect integrins clustered by engagement with extracellular matrix constituents to the cytoskeleton [52], [53], [54]. In addition to providing a physical anchor, focal adhesions serve as scaffolds for complex bidirectional signaling networks in control of
PIX and GIT: determinants of cell polarity and directional motility
Cell spreading is a suitable context for dissecting mechanisms of membrane ruffling and focal adhesion turnover, but it does not permit the study of the polarization events elicited by chemotactic, haptotactic, and cell–cell contact-mediated stimuli that govern directional cell migration. Several recent studies using widely different cell systems and experimental conditions support the conclusion that PIX and GIT molecules serve pivotal functions in specifying cell polarization and
Concluding remarks
The PIX–GIT complex regulates membrane ruffling, focal adhesion turnover, and polarity in motile cells, and consequently cell spreading as well as directional migration. These functions are executed irrespectively of the mechanisms by which the PIX and GIT are recruited to the membrane, thus suggesting that the complex serves as a cassette that can be utilized in a wide range of biological contexts. The examples given above undoubtedly underestimate the physiological significance of the PIX–GIT
Acknowledgements
We extend our apologies to investigators whose work was not included due to constraints on topic and space. Work in our laboratory is supported by the Roy and Lynne Frank Foundation and by R0-1 CA092354 from NIH (to SHH). We are grateful to Dr. Mirjam M.P. Zegers for sharing unpublished results.
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