Merck UK - Calbiochem: G-Proteins & Related Products

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G-Proteins & Related Products

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G-Proteins - An Introduction

The heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) mediate signaling from a large number of diverse seven transmembrane spanning cell surface receptors (7TMSRs) to a variety of intracellular effectors. These signal transduction pathways control numerous essential functions in all tissues and are ubiquitous throughout the animal kingdom. G-proteins are composed of a 36 - 52 kDa a-subunit, a 35 - 36 kDa b-subunit and an 8 - 10 kDa g-subunit. The b- and g-subunits are assembled into bg dimers that act as functional units which are only separable under strongly denaturing conditions. The a-subunits are capable of binding guanine nucleotides and are active when GTP is bound and inactive when GDP is bound. Early models of G-protein action assumed a direct and specific role for the a-subunits in modulating effector actions and a more passive role for the bg-subunits as attenuators of a activity. It is now clear that, in addition to controlling the response to mating factors in yeast, bg-subunits play a direct signaling role in modulating the activity of some potassium channels, adenylyl cyclases, and phospholipases in mammalian cells. The currently accepted mechanism of G-protein mediated signal transduction is summarized in Figure 1. Agonist binding to a 7TMSR alters the receptor’s conformation allowing it to catalyze the exchange of GDP for GTP on the a-subunit of the G-protein coupled to the receptor. The binding of GTP produces a conformational change in the G-protein a-subunit resulting in the dissociation of the receptor- G-protein complex and freeing the a- and bg-subunits to interact with their target effectors. The slow intrinsic GTPase activity of the a-subunit hydrolyzes the bound GTP to GDP, ending the interaction of the a-subunit with its effector. The GDP bound a-subunit interacts strongly with bg-subunits and thus GTP hydrolysis presumably also terminates bg-mediated signaling by promoting the association of the GDP-bound a-subunits with bg-dimers. Ultimately, signaling is controlled by these three interdependent association- dissociation cycles: interactions between receptor and G-proteins; between guanine nucleotides and Ga; and between Ga and Gbg. Thus, the first amplification of the signal occurs because a single agonist-occupied receptor is capable of activating multiple G-proteins and the duration of the signal is, in part, determined by the lifetime of the GTP-bound form of the a-subunit.

Among the most exciting recent developments in the G-protein field are determinations of the crystal structures of GDP and GTPgS liganded a-subunits, an isolated bg-dimer, and an intact abg-heterotrimer. Comparisons among these structures provide clear insights into the actual molecular mechanisms by which G-proteins accomplish their switching functions in signaling pathways. Not surprisingly, there are significant conformational differences between GDP and GTPgS liganded a-subunits. While some 86% of the amino acid residues occupy identical positions in the two structures, the 14% of the residues that change positions are involved in conformational changes that bring three “switch regions” that lie apart from one another in the GDP bound a-subunit into close apposition along a common face of the GTPgS bound subunit. Remarkably, the change between these conformations is controlled by the g-phosphate of a GTP molecule which contacts just three amino acids. The realization that the b-subunit is shaped like a seven-bladed propeller, highly similar to several other proteins without obvious sequence (other than a highly conserved WD repeat forming the propeller blades) or functional similarities, is one of the major surprises revealed by the crystal structures. Another surprise is the lack of structural differences between an isolated bg-dimer and the bg-dimer of an intact heterotrimer. Apparently, these propeller structures form quite rigid scaffolds and the absence of conformational change seems to be a family characteristic. The fact that the g-subunit lies along the side and bottom of the b propeller structure making very few contacts with itself explains why it may only be removed from its b-subunit partner under strongly denaturing conditions. The present view is that the bg dimer serves as a rigid lever prying open the guanine nucleotide-binding pocket of the a-subunit in response to receptor activation.

The components of G-protein coupled signaling pathways, the receptors, the G-proteins, and the effectors, may be classified into major families on the basis of structural and functional criteria. The abundance of distinct subtypes identified within the major families of these components is a striking feature of these signaling pathways. The G-proteins are usually classified by the nature of their a-subunit, with 23 distinct a-subunits encoded by 17 different genes grouped into four general classes (a_s, a_i/o, a_q, and a₁₂) based on amino acid similarities. Most of the a-subunits are widely distributed across tissue types with the exception of the a-subunits mediating sensory transduction (vision, taste, and olfaction). Unfortunately, the functional role of specific a-subunits is not defined by their structural classifications. While there is some general discrimination among the major families of a-subunits for receptors and effectors, understanding the mechanisms that underlie signaling selectivity remains an important unanswered question. It is clear that, various a-subunits are capable of interacting with the same effectors, that individual a-subunits are capable of modulating different effectors, and that individual receptors are capable of interacting with different a-subunits. Indeed, with the observation that human thyrotropin receptors interact functionally with members from each of the four major families of G-protein a-subunits, extreme caution should be adopted when generalizing about selectivity in these signaling pathways.

The functional properties of the a-subunits are, in part, determined by the covalent attachment of various lipids. Reversible palmitoylation of an N-terminal cysteine has been described for all a-subunits except transducin, while the irreversible myristoylation of an N-terminal glycine is restricted to members of the a_i/o family. Although the precise functional significance of these modifications is unknown, they appear to facilitate membrane association and functional interactions with other components of the signal transduction systems, especially the bg-subunits and effector molecules. Several of the a-subunits are also substrates for covalent modification by certain bacterial toxins. Cholera toxin is capable of ADP-ribosylating members of the a_s family, inhibiting their ability to hydrolyze GTP and leaving them constituitively activated. Pertussis toxin ADP-ribosylates some, but not all, members of the a_i/o family, uncoupling them from their cognate receptors and thereby inhibiting signaling mediated by such receptors. These toxins have been invaluable in identifying many G-protein-mediated responses. Another family of compounds, the so-called receptor mimetics, including mastoparan and melittin, have been useful in studying G-protein mechanisms because of their ability to directly activate many a-subunits.

A similar diversity is exhibited by the G-protein bg-subunits, with five b and twelve g-subunits now described. Interestingly, not all the possible pairs of b- and g-subunits can be formed. Most of the b- and g-subunits are ubiquitously expressed (albeit at varying levels), with the exceptions of g1, which is restricted to retinal rods, and b3, which is preferentially expressed in retinal cones. The g-subunits contain a “CAAX” box that directs their complex C-terminal processing which results in the proteolytic removal of three amino acids and covalent attachment of either C15 or C20 iso-prenyl groups and carboxymethylation of the C-terminal cysteine. These lipid modifications have been shown to be important for interactions with membranes and the other components of the signaling pathways. In addition to their role in the direct modulation of effector molecules described above, bg-subunits are involved in the targeting of specific G-protein receptor kinases (GRKs) responsible for the desensitization of many receptor signals. Recently, the bg-subunits have been shown to participate in the activation of mitogen activated protein kinases (MAPKs) initiated by some 7TMSRs. Interest in the G-protein bg-subunits continues to increase and it is quite likely that additional signaling roles for G-protein bg-subunits remain to be elucidated.

While much is now known about these remarkable signaling pathways, many questions persist and exciting new areas of investigation are emerging. Although GTP hydrolysis by the a-subunit is considered to be the mechanism for ending signaling along G-protein regulated pathways, it has been known for some time that this reaction occurs too slowly to account for the rapidity of many physiological responses. A related family of signaling molecules, the low molecular-weight G-proteins typified by Ras, utilizes a large array of accessory proteins to regulate guanine nucleotide exchange and hydrolysis. Thus, much excitement was generated by the discoveries that cGMP phosphodiesterase and PLC-b, two effectors regulated by G-protein a-subunits, were capable of accelerating the rate of GTP hydrolysis by such a-subunits. Recently, genetic techniques have described a novel class of regulators of G-protein signaling (RGS) proteins. Two members of the RGS family have been shown to accelerate the rate of GTP hydrolysis by a_i-subunits but not by a_s subunits and, therefore, these proteins represent a whole new realm for selective regulation of G-protein signaling pathways. Other important areas for investigation involve understanding the mechanisms by which the G-protein signaling cascades are intertwined with the growth and differentiation pathways primarily characterized with cytokine and growth factor receptors.

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G-Protein Activators

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Antibodies to G-protein Subunits & small GTP-Binding Proteins

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G-Protein Subunits, small GTP-Binding Proteins & Other

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