By Thomas G. Brock, Ph.D.

At the cell surface, there are receptors that couple with enzymes, like the receptor tyrosine kinases, and there are those that activate transmembrane ion channels. A third family of receptors includes the G protein-coupled receptors (GPCR). These receptors, also known as seven transmembrane receptors because each has seven regions that pass through the plasma membrane, constitute a large family of literally hundreds of human members.¹ Different GPCRs respond to such diverse stimuli as light, volatile compounds, bioactive lipids, cytokines, hormones, and neurotransmitters. Receptor activation puts specific, dedicated G proteins into play, which in turn alters the activity of enzymatic signaling pathways. This article presents different aspects of GPCR action.

Signaling Through the Heterotrimeric G Protein

Signaling begins with activation of the GPCR, typically by binding of an appropriate ligand to a specific ligand binding domain. This domain can be extracellular, as in the metabotropic glutamate receptors, or entrenched in the plasma membrane, as in the GPCRs for the bioactive lipid leukotriene B₄. An interesting group includes the opsins, light-sensitive GPCRs found in photoreceptors of the retina. The opsins themselves are the seven transmembrane proteins, each of which covalently binds a vitamin A-based chromophore linked to a lysine residue in the seventh transmembrane region, within the membrane itself. The absorption of a photon of light causes isomerization of the chromophore, resulting in a conformational change in the opsin protein, just as ligand binding does to other GPCRs. The GPCR can now activate a trimeric GTP-binding protein, or G protein (Figure 1).

Figure 1. G protein activation

The G protein is composed of three protein subunits, Gα, Gβ, and Gγ. Both the Gα and Gγ subunits are post-translationally modified to have covalently attached lipid tails, which anchor the G protein to the plasma membrane. In the resting, unstimulated state, the Gα subunit contains GDP and the G protein is inactive. The inactive G protein may be associated with an inactive receptor or it may only bind after the receptor is activated. In both situations, the activated receptor acts as a guanine nucleotide exchange factor (GEF), inducing the release of GDP from the G protein. GTP, which is abundant in the cytoplasm, replaces the GDP, activating both the Gα subunit and the β/γ complex. In some cases, the activated Gα subunit separates from the β/γ complex, whereas, in other cases, the two activated components remain together. In either case, both of the activated components can now regulate the activity of target proteins in the plasma membrane, including adenylate cyclases, phospholipase C (PLC) isoforms, potassium and calcium ion channels, guanine-nucleotide exchange factors for the GTPase Rho A (RhoGEFs), and other effector enzymes. The activated target proteins then propagate the signal forward to other components in the signaling cascade.

The Gα subunit, in addition to being an intermediary in activating a target protein, is a GTPase, ultimately hydrolyzing its bound GTP to GDP, thus inactivating itself. This step can be accelerated by the binding of another protein, called regulators of G protein signaling (RGS), or, more accurately, GTPase-accelerating proteins (GAP).² The inactivated GDP-bound Gα subunit actively recruits specific β/γ complexes, stopping their signaling to re-form an inactive G protein. This G protein can once again interact with the activated receptor to repeat GDP/GTP exchange and signal propagation. Interestingly, some receptors fastidiously activate specific types of G proteins, while other receptors may be described as ‘biased' toward a certain G protein or even ‘promiscuous' in G protein activation. Ultimately, activated receptors are phosphorylated on cytoplasmic residues by GPCR kinases (GRKs). Phosphorylation promotes high-affinity binding with an arrestin protein, preventing further interaction of the receptor with G proteins. Arrestin binding also targets the receptor for internalization via clathrin-coated pits.

Two Related Examples

The hundreds of unique GPCRs, with their thousands of different ligands, all funnel their actions through G proteins, with the critical component typically being the Gα subunit. As an example, many GPCRs signal exclusively through Gαs, which stimulates the activity of certain forms of adenylate cyclase (ADCY). The ADCY constitute a family of proteins which synthesize cAMP from ATP (Figure 2). If cAMP is not metabolized by phosphodiesterases (PDE), then it can activate two pathways: protein kinase A (PKA) and exchange proteins activated by cAMP (Epac). In resting cells, PKA exists as a tetramer of two regulatory subunits holding two catalytic subunits in an inactive state. The association of cAMP with the regulatory components causes dissociation of the tetramer, allowing the free, active catalytic subunits of the kinase to phosphorylate target proteins.³ Perhaps most notably, PKA phosphorylates CREB, which binds the cAMP response element (CRE) and alters gene transcription. PKA can also target other transcription factors (e.g., NF-κB, NFAT, RARα), as well as a wide variety of other proteins (e.g., BAD, PLCγ1, histone H3). Interestingly, PKA also phosphorylates inhibitors of protein phosphatases PP₁ and PP2A, preventing the dephosphorylation of PKA and non-PKA targets. The Epac proteins represent 2 of 6 human Rap GEFs. By replacing GTP for GDP in Rap, Epacs modulate kinase signaling, mitogenesis, and exocytosis.⁴

Figure 2. Signaling through a Gα_s GPCR, the prostaglandin E₂ receptor EP₂, and a Gα_i GPCR, the metabotropic glutamate receptor mGluR3, to adenylate cyclase (ADCY)

On the flip side of this stimulatory pathway is GPCR signaling through an inhibitory subunit, Gα_i. Activation of a GPCR that puts Gα_i in play suppresses the generation of cAMP by ADCY. This can be important in certain cell types that have constitutive activation of ADCY and high basal levels of cAMP. More commonly, Gα_i-mediated inactivation of ADCY blocks concomitant or subsequent attempts to activate ADCY through Gα_s-dependent GPCRs. For some reason, the Gβγ dimers which dissociate from Gα_i are particularly involved in activating other signaling cascades. Thus, as Gα_i is inhibiting cAMP production, its G protein partners may activate certain isoforms of PLC, phosphoinositide 3-kinase, and select ion channels, like the G-protein-regulated inward rectifier K⁺ channels. The diversity of effects of different G protein subunits suggests a closer look at each of the distinct subunits is warranted.

G Protein Subunits

In the annotated database UniProtKB/Swiss-Prot, the Gα subunits are grouped into four subfamilies: Gα_s, Gα_i/o/t/z, Gα_q, and Gα₁₂. All Gα subunits have four GTP binding sites and hydrolyze GTP. They are typically N-terminally myristoylated or palmitoylated, which is necessary for membrane association. Most of the 17 human Gα subunits consist of 350-395 aa organized in a globular structure dominated with α helices (Figure 3). A single gene is the source for two major Gα_s isoforms, Gα_s1 and Gα_s2, through alternative splicing; an additional extra long (XLas) isoform of 1037 aa is also derived from the same gene. A distinct gene gives rise to an olfactory type Gα_s subunit (Golf), which is expressed on chemosensory organs of many organisms.⁵ As noted above, G proteins containing the Gα_s subunit drives cAMP generation by ADCY. All of the Gα_s isoforms can also be activated by cholera toxin, which induces ADP-ribosylation on a key arginine residue resulting in constitutive activation.

Figure 3. The structure of the heterotrimeric G protein, with GDP bound to Gα (cyan) and Gβ (magenta) linking Gα to Gγ (green)⁸

The Gα_i/o/t/z group includes eight members closely related by structure if not function. As outlined above, the three Gα_i members would be expected to inhibit adenylate cyclase, which Gα_i1 and Gα_i2 do. However, Gα_i3, which also goes by the name Gα_k, stimulates receptor-regulated K⁺ channels, even though its sequence is a 98% positive match with Gα_i1. Two of the three Gα_t, elements of the transducin G protein subunits, couple activation of the rhodopsin receptor by visual impulses with cGMP PDE; Gα_t1 acts in rods and Gα_t2 is in cones.⁶ Gα_t3, a component of gustducin, links bitter, sweet, and umami taste sensation with cGMP PDE stimulation. The Gα_o and Gα_z subunits are involved in diverse receptor pathways but their modes of signaling are obscure. All of these family members, except Gα_z, are ADP-ribosylated on cysteine residues, and inhibited, by pertussis toxin.

The Gα_q subfamily activates phospholipase C (PLC), which converts phosphatidylinositol 4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). IP₃ induces the release of calcium from intracellular stores, propagating Ca²⁺-dependent signaling. Also, Ca²⁺ acts with DAG to turn on certain isoforms of PKC. This family, also referred to as Gα_q/11, includes Gα₁₄ and Gα₁₅, as well as Gα_q and Gα₁₁. Gα₁₅ is specifically expressed in hematopoietic cells. Finally, the Gα₁₂ subfamily has two members, 12 and 13. Both interact with UBX domain-containing protein 11 (UBXN11) to promote the Ras homolog RhoA, a small GTPase that regulates actin reorganization. In addition, Gα₁₃ binds the integrin α11bβ3 to modulate ligand-integrin signaling through RhoA.⁷

Less is known about the Gβ and Gγ subunits. There are 5 human Gβ proteins of 340-295 aa. Each contains 7 WD repeats, which facilitate forming the G protein trimer (Figure 3). The 12 human Gγ proteins are small, only 67-75 aa. Gγ_T1 and Gγ_T2 are restricted to transducins in retinal rods and cones, respectively, while Gγ₇ associates with Golf. All are a subject of current research.

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