How do cells communicate with one another and sense and respond to their environments? Cells of eukaryotic organisms utilize a suite of signaling components that, together, form heterotrimeric guanine nucleotide binding protein (G protein) complexes. In the classical G protein signaling model, this complex is formed by an inactive seven-pass transmembrane G protein coupled receptor (GPCR) that is bound on the cytosolic surface of the plasma membrane to an inactive heterotrimeric G protein. This G protein consists of an inactive GDP-bound Gα subunit, a Gβ subunit, and a Gγ subunit. Both Gα and Gγ are tethered to the plasma membrane via covalent lipid modifications.
G protein signaling begins when a stimulus activates the GPCR, causing a conformational change in the cytoplasmic domains of the receptor. The guanine-nucleotide exchange factor activity of the GPCR induces a conformational change in the bound Gα-GDP, allowing Gα to release GDP. Because GTP is in excess in the cell, Gα readily binds GTP, activating the molecule. This activation allows Gα to dissociate from the Gβγ heterodimer (which remain tightly bound throughout the signaling process and require one another for protein stability), and exposes the shared binding surfaces. These surfaces, along with other solvent-exposed surfaces on the proteins, serve as sites of protein-protein interactions between each subunit and its downstream effectors. By binding to downstream effectors, Gα and Gβγ propagate signaling cascades. This signaling is terminated when the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP and the inactive heterotrimer reforms. In addition to the Gα GTPase, accessory proteins such as those belonging to the regulator of G-protein signaling (RGS) family can accelerate this hydrolysis.
Fig. 1 Canonical heterotrimeric G protein signaling. Inactive and active G protein complexes are shown as discussed in the text.
Of course, G protein signaling is not as simple as this ideal paradigm suggests. Although the theme remains constant, many variations of this signaling regime have been identified. For instance, mammalian systems contain 23 Gα subunits, five Gβs, and twelve Gγs in addition to hundreds of GPCRs. Dozens of downstream effectors of both Gβγ and Gα have been identified in addition to a number of scaffolding and regulatory molecules. The suite of components in any given cell is a product of the individual organism and the specific cell type. However, it has been shown that many components of the heterotrimer can bind promiscuously, such that several different combinations of heterotrimer subunit proteins or Gβγ dimers are possible. These different subunit combinations enlarge the signaling capabilities of this complex while also providing signaling specificity.
G protein components have been grouped into families based on their known function, sequence homology, and evolutionary history. Gα proteins can be grouped into four main classes (G(io), G(q), G(s), and G(12)), while Gβ proteins can be grouped into two classes (Gβ1-like and Gβ5-like). For both molecules, the evolution that followed rounds of duplication was constrained in part by residues on the molecules’ surfaces that are involved in protein-protein interactions with effectors, regulators, or other G protein components. The primordial G protein components duplicated and diverged throughout evolutionary history, although the plant G protein components contain the most characteristics that are similar to the common ancestor.
Activated G protein subunits positively and negatively regulate downstream effector activity. Although researchers once believed that the sole functions of Gβ was to inhibit Gα signaling and to localize Gα to the membrane, it is now clear that Gβ regulates downstream signaling cascades via interactions with a variety of effectors and accessory proteins. Gβ proteins have been studied extensively in mammalian systems; multiple mammalian effectors have already been identified. Gβ stimulates G protein-gated inwardly rectifying potassium channels (GIRKs), which regulate membrane potential in a cell-type dependent manner. Gβ activates phospholipase C β, ultimately resulting in the release of Ca2+ into the cytoplasm. In neurons, Gβ inhibits the activity of N-type Ca2+ channels, affecting the release of neurotransmitters. Mammalian accessory proteins that interact with Gβ have also been identified. Retinal phosducin regulates G protein subunit availability, possibly by assisting in the proper localization Gβ or by sequestering Gβ in the cytoplasm; this regulation facilitates the adaptation to light. Additional proteins with similarity to phosducin are expressed more ubiquitously than retinal rhodopsin and may play function as signaling molecules or localization chaperones. The RGS R7 family of proteins binds preferentially to Gβ5-like proteins via a G-gamma-like (GGL) domain on RGS. The RGS GGL domain binding to Gβ precludes the binding of Gγ, resulting in a Gβ-RGS heterodimer instead the traditional Gβγ heterodimer. This is the only known example whereby Gβ is stabilized by a protein other than Gγ.
The three-dimensional structure of Gβ provides insights into its function and regulation of downstream effectors. Gβ is formed by an N-terminal alpha helix of approximately twenty residues, which interacts with Gγ, and a C-terminal seven-bladed beta-propeller that contains seven WD repeat sequences. When inactive, the “top” surface of this beta propeller structure is occupied by the binding of Gα; upon activation, no conformational change occurs on the Gβ molecule, but this binding surface is exposed and is highly utilized to form a portion of the binding interfaces between Gβ and its downstream effectors. Thus, the interactions between Gβ and its effectors are regulated by its binding site availability. Within this binding region, several key residues termed “hot spots” form critical contacts between Gβ and its effectors.