BIO254:Gprotein

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. 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).

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). Figure 1 schematizes the switch mechanism for the Rho protein.

Figure 1. Image taken from Luo, 2000.

Heterotrimeric G proteins are unique in that they exist as a complex (G_αβγ) in the GDP-bound state but dissociate (into G_α and G_βγ) upon the release of GDP/binding of GTP.

Heterotrimeric G proteins

Heterotrimeric G proteins consist of three subunits: α, β and γ. The alpha subunit harbours the GDP/GTP binding site and the GTPase activity of the G-protein (Fig. 2) The C-terminus of the α subunit makes the G protein bind to the cytosolic side of specific membrane-bound GPCRs (see below).

Figure 2. 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 the α subunit to 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. 3a), 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. 3b).

Figure 3a. Image modified from http://nobelprize.org/nobel_prizes/medicine/laureates/1994/illpres/disc-gprot.html.

Figure 3b. Image modified from http://nobelprize.org/nobel_prizes/medicine/laureates/1994/illpres/disc-gprot.html.

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). The association of the receptor with all three G protein subunits, G_α, G_β, and G_γ, requires that GDP is bound to G_α. When the receptor protein 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 proteins then dissociate from the receptor and from each other, with only the β- and γ-subunits remaining bound to one another. G_βγ and G_α-GTP may then activate downstream effectors. Figure 4 is a schematic of this dissociation. G_α-GTP is shown activating adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP). cAMP is an important second messenger.

Figure 4. Image modified from Firestein, 2001.

For more information on G protein-coupled receptors, see the GPCR wikipedia entry

Families

Heterotrimeric G proteins have been divided into four families on the basis of sequence similarity: G_s, G_i, G_q, and G_12/13. These four families have been shown to have different, but often overlapping, effects on the cell (see figure 3, Neves, 2002).

Figure 3. Image taken from Neves, 2002

The original GPCR cell signaling pathway described included G_s proteins. Gα_s, among other things, activates adenylate cyclase. G_i pathways are characterized by the ability of Gα_i to inhibit adenylate cyclase, and of G_βγ to activate its own downstream effectors, which include phosphatidylinositol 3-kinase (PI3K). The G_q pathway is activated by calcium-mobilizing hormones and acts through inositol trisphosphate (IP3), diacylglycerol (DAG), and protein kinase C (PKC). The G_12/13 family is the most recently identified and the least well studied. It is not known as to what extent Gα₁₂ and Gα₁₃ act through distinct effectors (Neves, 2002).

The Rho/Rac family of small GTPases

The Rho family of small G proteins, which includes Rho, Rac, and CDC42, are important effectors that regulate 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 4 describes the relationship between Rho, Rac, CDC42, Rho GEF/GAPs, and actin (Huber, 2003).

Figure 4. Image taken from Huber, 2003

References

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

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

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

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

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

6. 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)

External Links

• GPCR wikipedia entry
• G protein wikipedia entry

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