BIO254:Gprotein

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Introduction

The term G protein refers to proteins that bind the nucleotide guanine as guanosine triphosphate (GTP) and guanosine diphosphate (GDP). There are two types of G proteins: heterotrimeric, or large, G proteins and small G proteins. Heterotrimeric G proteins are membrane-associated and, along with G protein-coupled receptors (GPCRs), function primarily in cell signalling and signal transduction. Small GTP-binding proteins function in diverse cellular processes including signal transduction, cytoskeletal reorganization, and vesicle trafficking (Takai et al., 2001).

A molecular switch

G protein activity is dependent on whether it is binding GTP or GDP. This useful property has led to the appropriation of G proteins by many cellular processes to be used as "molecular switches". G proteins are generally thought to be "active" when binding GTP and "inactive" when binding GDP. The transition from the GTP-bound state to the GDP-bound state depends on the hydrolysis of GTP. This GTPase activity is either completely intrinsic to the G protein or is enhanced by another class of proteins, "GTPase activating proteins" (GAPs). The GDP to GTP transition requires the dissociation of GDP, so that GTP may again bind at the active site. Proteins that mediate this GDP dissociation are known as guanine nucleotide exchange factors (GEFs).

Heterotrimeric G proteins

Heterotrimeric G proteins are comprised of three subunits -- α, β and γ -- that exist as a complex (Gαβγ) in the GDP-bound state but dissociate (into Gα and Gβγ) upon the release of GDP and binding of GTP. Gα contains the GDP/GTP binding site and GTPase activity (Fig. 1). The C-terminus of Gα gives the G protein affinity for specific membrane-bound GPCRs (see below).
Figure 1. Image from (Milligan, 2006).  Some parts have been removed for clarity.  GDP is shown in purple.  The α subunit C-terminus residues, shown in blue, convey GPCR specificity.  The N-terminus helix, shown in red, is required for binding of Gαto the other subunits.
Figure 1. Image from (Milligan, 2006). Some parts have been removed for clarity. GDP is shown in purple. The α subunit C-terminus residues, shown in blue, convey GPCR specificity. The N-terminus helix, shown in red, is required for binding of Gαto the other subunits.

Discovery

The role of cAMP-dependent signal transduction was known in the 1950s and 1960s; however, the essential role of GTP was masked by the fact that cAMP preparations were contaminated by GTP (Milligan, 2006). In the 1970s a mutated cell line was found to have an intact ligand receptor and amplifier, yet this cell line did not respond to the receptors ligand (Fig. 2a), implying the existence of an intermediary and also providing a cell line on which reconstitution assays could be performed. Alfred G. Gilman purified and identified this intermediary in 1980 (Northup, 1980) by reconstituting the complete pathway by adding a purified protein, the G-protein (Fig. 2b).

The heterotrimeric G protein that Gilman isolated increased cAMP levels. In 1980 Martin Rodbell wrote a review (Rodbell, 1980) that helped direct the search for the first cAMP reducing G-protein to be discovered, in 1984. Martin Rodbell and Alfred G. Gilman were awarded the 1994 Nobel Prize in Physiology and Medicine for the discovery of "G-proteins and the role of these proteins in signal transduction in cells". Since the first G-proteins were identified, many others with effectors other than cAMP have been cloned, in many cases by homology. Currently 16 alpha, 5 beta, and 14 gamma subunits have been identified (Milligan, 2006).

G Protein-Coupled Receptors

Heterotrimeric G proteins associate with 7-transmembrane domain receptors called G protein-coupled receptors (GPCRs) at the cell membrane. There are as many as 865 GPCR-encoding genes in humans (Milligan, 2006). Specific GPCRs are recognized by specific G proteins. This recognition is mediated by a sequence at the C-terminus of the G-protein α subunit. For more information on G protein-coupled receptors, see the GPCR wikipedia entry

Mechanism

Binding to GDP increases the affinity of a G protein for its GPCR. When a G-protein-bound GPCR is activated with the appropriate ligand, the ligand/receptor complex acts as a GEF, allowing the GDP to dissociate and GTP to bind. The G protein then dissociates from the GPCR and the α separates from the β- and γ-subunits which remain bound to one another. Gβγ and Gα-GTP may then activate downstream effectors. Figure 3 is a schematic of this dissociation, specifically for the case of a G-protein with adenylate cyclase as its effector; there are G-proteins with many other different types of effectors (see below). Gα-GTP is shown activating adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP), an important second messenger.
Figure 3. Image modified from Firestein, 2001.
Figure 3. Image modified from Firestein, 2001.
The rate of conversion of GTP to GDP is modulated by GEFs such as those of the RGS family as illustrated in Figure 4.
Figure 4. Image from (Milligan 2006).
Figure 4. Image from (Milligan 2006).

Families

Heterotrimeric G proteins have been divided into four families on the basis of sequence similarity: Gs, Gi, Gq, and G12/13. These four families have been shown to have different, but often overlapping, effects on the cell (Fig. 5) (Neves, 2002).
Figure 5. Image taken from Neves, 2002
Figure 5. Image taken from Neves, 2002

Effectors

Heterotrimeric G proteins act through a large range of effectors (Table 1).
Table 1. Effectors, expression patterns of heterotrimeric G proteins.  Taken from (Milligan, 2006)
Table 1. Effectors, expression patterns of heterotrimeric G proteins. Taken from (Milligan, 2006)

The original GPCR cell signaling pathway described was a Gs protein that activates adenylate cyclase. Certain Gi pathways are characterized by the ability of Gαi to inhibit adenylate cyclase. Gβγ subunits have their own downstream effectors, which include phosphatidylinositol 3-kinase (PI3K). Certain Gq pathways act through inositol trisphosphate (IP3), diacylglycerol (DAG), and protein kinase C (PKC). The Gα12 and Gα13 family effectors include phospholipases.

Roles in the Nervous System

Many heterotrimeric G proteins are specific to certain cell types and tissues (Table 1). Certain heterotrimeric G proteins are expressed specifically in nervous system components including olfactory neurons, CNS ganglia, neuroendocrine cells, astroglia, and retinal rod and cone cells. In the nervous system heterotrimeric G proteins are found in signaling pathways mediated by dopamine, epinephrine, serotonin, glucagon, light, olfactory signals, and other factors. They are involved in taste, vision, affect, arousal, and other functions.

Small G Proteins

In contrast to heterotrimeric G proteins, small G proteins are monomeric. Small G proteins are between 25 and 40 kD, which is indeed smaller than the heterotrimeric G proteins, the alpha subunit of which alone is 45 kD. There are over 100 small G proteins. The small G protein superfamily includes the Ras family (signal transduction), the Rho/Rac family (cytoskeleton), the Rab and Sar1/Arf families (vescicle trafficking), and the Ran family (nuclear import/export) (Takai et al., 2001). Like heterotrimeric G proteins, small G protein function involves activation by binding to GTP and release of GDP and inactivation by hydrolyis of GTP to GDP. The kinetics of these steps are such that small G proteins can and do act as biological timers. The mechanism of small G protein function is demonstrated in Figure 6 for the case of the Rho protein.
Figure 6.  Image taken from Luo, 2000.
Figure 6. Image taken from Luo, 2000.

The Rho/Rac family of small GTPases

The Rho family of small G proteins, which includes Rho, Rac, and CDC42, are important regulators of actin dynamics. These proteins are of particular importance at the growth cone, where they mediate growth and collapse in response to chemoattractants and repellents. Axon guidance receptors are directly or indirectly coupled to Rho GEFs and GAPs, which regulate Rho activity. Figure 7 describes the relationship between Rho, Rac, CDC42, Rho GEF/GAPs, and actin (Huber, 2003).
Figure 7. Image taken from Huber, 2003
Figure 7. Image taken from Huber, 2003

References

1. Takai Y, Sasaki T, Matozaki T. Small GTP-Binding Proteins. Physiol Rev. 81, 153-208 (2001).

2. Milligan G, Kostenis E. Heterotrimeric G-proteins: a short history. Br J Pharmacol. 147 Suppl 1:S46-55 (2006)

3. Northup JK, Sternweis PC, Smigel MD, Schleifer LS, Ross EM, Gilman AG. Purification of the regulatory component of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 77 6516–6520 (1980).

4. Rodbell, M. The role of hormone receptors and GTP regulatory proteins in membrane transduction. Nature 284, 17–22 (1980).

5. Firestein, S. How the olfactory system makes sense of scents. Nature 413, 211-218 (2001)

6. Neves S, Ram P, Iyengar R. G protein pathways. Science 296, 1636-1639 (2002)

7. Luo L. Rho GTPases in neuronal morphogenesis Nat Rev Neurosci. 1, 173-180 (2000).

8. Huber A, Kolodkin A, Ginty D, Cloutier JF. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Ann Rv Neurosci 26, 509-63 (2003)

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