Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

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Abstract

Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gβγ subunits.

Introduction

Signal processing via heterotrimeric G-proteins generally involves an initial input sensed by a cell surface G-protein coupled receptor (GPCR), leading to conformational changes in receptor subdomains transferring this signal to a G-protein, promoting the exchange of guanosine-5′-triphosphate (GTP) for guanosine-5′-diphosphate (GDP) and subunit dissociation or rearrangement, which allows Gα and Gβγ to regulate a number of downstream signaling molecules. Multiple discoveries over the last several years have forced us to broaden our perspective on the role of G-proteins as “signaling switches”, recognizing that (1) these entities are regulating intracellular events independent of their roles as transducers for GPCR; (2) the Gα and Gβγ subunits may function independently of each other; and (3) the existence of accessory proteins that provide unexpected modes of signal input in this context.

The discovery of alternative modes of regulation of G-proteins and unexpected functional roles for these proteins resulted from a confluence of several independent lines of investigation. A biochemical approach built upon data suggesting cell-specific differences in signal transfer from R to G, the partial purification of a putative nonreceptor G-protein activator from extracts of NG108-15 cells, and the identification of other nonreceptor proteins that could influence the activation state of G-proteins (see Cismowski & Lanier, 2005). An extension of this line of investigation led to the development of a functional yeast-based screen for mammalian entities that activated G-protein signaling in the absence of a receptor (Cismowski et al., 1999, Takesono et al., 1999). In parallel with these studies were the initiatives of several laboratories to search for Gα and Gβγ binding partners in yeast 2-hybrid screens (see Table 3 in Sato et al., 2006a) and on the realization that G-protein subunits were associated with intracellular organelles (Stow et al., 1991, Wilson et al., 1994). Interspersed with these biochemical approaches was the realization that there were changes in signal processing through G-protein signaling systems that occurred independent of any obvious changes in receptor number or G-protein expression levels, suggesting additional undefined regulatory mechanisms.

Another line of investigation evolved out of the study of asymmetric cell division in Drosophila melanogaster neuroblasts and sensory organ precursor cells in parallel with the C. elegans embryo. Gotta and Ahringer (2001) reported that Gβγ regulated the orientation of the mitotic spindle in C. elegans in the 1-cell embryo. In addition, Gα and a protein containing a G-protein regulatory (GPR) motif (a signature feature of Group II AGS proteins discussed in detail later in this review) interacting with Gα were identified as regulators of asymmetric cell division in a large scale RNAi-based functional screen (Kamath et al., 2003). Parallel studies in D. melanogaster also led to the identification of key players involved in this biological process (Betschinger & Knoblich, 2004). One of these key players was partner of inscuteable (Pins), which contains 4 GPR motifs and is an ortholog of the Group II AGS proteins AGS3 and LGN (discussed later in this review). Pins and its binding partner, Inscuteable (Insc), influence the positioning of cell fate determinants to generate asymmetry and are important for the stability and targeting of protein complexes that transmit polarity information at the apical cortex of the neuroblast to orient the mitotic spindle. Notably, Pins was identified as a binding partner of Gα (Parmentier et al., 2000, Schaefer et al., 2000, Yu et al., 2000, Bellaiche et al., 2001, Schaefer et al., 2001).

Thus, we had the confluence of biochemical data indicating unexpected modes of regulation for heterotrimeric G-proteins and data in model organisms implicating Gα and Gβγ in control of asymmetric cell division. One of the many interesting aspects of the signaling role played by G-proteins in the asymmetric cell division in the model organisms was that the process was apparently an intrinsically regulated event independent of a cell surface receptor. This initiated a lot of discussion in the literature about the implications of such a functional role for G-proteins, how this process is regulated and what G-proteins might be involved with in the cell independent of their well-characterized roles as transducers from cell surface GPCRs.

The investigations alluded to above revealed 4 major points that altered our basic concepts of G-protein signaling: (1) Gα and Gβγ are processing signals within the cell distinct from their role as transducers for cell surface receptors; (2) such signals involve previously unrecognized functional roles for heterotrimeric G-protein subunits; (3) Gα and Gβγ may exist complexed with alternative binding partners independent of the classical Gαβγ heterotrimer; and (4) the G-protein activation/deactivation cycle may be regulated, independent of nucleotide exchange.

This review focuses on the group of proteins defined in a yeast-based functional screen as receptor-independent activators of G-protein signaling (AGS) proteins. The goal of the review is to highlight concepts evolving from the discovery of alternative modes of G-protein regulation via AGS proteins, to discuss various unresolved issues in the field and to provide information on the current status of our knowledge regarding functional roles of AGS proteins. The reader is referred to other reviews for a broader discussion of additional G-protein regulators and a more detailed discussion of the discovery of AGS proteins and their initial characterization along with a more extensive listing of citations (Blumer et al., 2005, Cismowski and Lanier, 2005, Sato et al., 2006a).

Section snippets

Strategy to identify receptor-independent activators of G-proteins

A series of experimental approaches were developed in our laboratory and others to identify novel proteins that directly regulate the activation state of G-proteins. One approach involved the influence of cell and tissue extracts on nucleotide binding to purified G-proteins, and another approach utilized a yeast-based functional screen (Cismowski et al., 1999, Takesono et al., 1999, Cismowski et al., 2002, Ribas et al., 2002a). The first approach resulted in the partial purification and

Group I AGS proteins

AGS1 was first discovered as a dexamethasone-inducible, ras-related cDNA in AtT20 cells and termed DexRas (Kemppainen & Behrend, 1998) and subsequently named RASD1 by the HUGO Gene Nomenclature Committee. AGS1 was also identified in yeast 2-hybrid screens (Tu and Wu, 1999, Fang et al., 2000) and as a regulated cDNA in various other “discovery” platforms (Table 2). Among the superfamily of small G-proteins, AGS1 is most closely related (∼ 60%) to Ras homologue enriched in striatum (Rhes)/tumor

Group II AGS proteins

Group II AGS proteins are defined by the presence of at least 1 GPR motif, a 20–25 amino acid cassette that serves as a docking site for Gi/oα-GDP and Gtα (Fig. 2, Fig. 5) (Takesono et al., 1999, Peterson et al., 2000, Bernard et al., 2001, Blumer et al., 2005, Sato et al., 2006a).3 The GPR motif is also found in Purkinje cell protein-2 (Pcp2)/L7, RGS14, Rap1GapII and WAVE1 (see Blumer et al., 2005, Sato et al., 2006a

Group III AGS proteins

As discussed earlier in the yeast-based functional assay, Group II and Group III AGS proteins are both active in the G204AGiα2 background and are not antagonized by overexpression of RGS5, which accelerates signal termination. However, Group II proteins bind Gα, whereas the Group III proteins AGS2 and AGS8 bind Gβγ. AGS10 or Goα also obviously binds Gβγ. However, the mechanisms by which the Group III AGS proteins activate G-protein signaling in the yeast functional screen and function in

AGS and related proteins in signal adaptation and disease

The regulatory mechanisms operational with AGS proteins reveal unexpected diversity in the “G-switch” signaling mechanism and cellular functions regulated by G-proteins. This flurry of activity over the last 5–6 years has opened up new avenues for therapeutic manipulation of G-protein signaling and the expanded role that G-proteins may play in disease or tissue adaptation. Many questions remain to be addressed including the following. What controls the activity of AGS proteins? Are Gα and Gβγ

Acknowledgments

This work was supported by MH90531 (SML), NS24821 (SML), GM074247 (SML), F32MH65092 (JBB), GM053536 (AVS) and GM060286 (AVS) from the National Institutes of Health. SML is greatly appreciative for this support and that provided by the David R. Bethune/Lederle Laboratories Professorship in Pharmacology and the Research Scholar Award from Yamanouchi Pharmaceutical Company, LTD (Astellas Pharma). SML also appreciates the sustained collaboration with Drs. Emir Duzic, Mary Cismowski and John

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