The GTP-binding protein (G protein) is a heterotrimeric protein composed of an α-, a β- and a γ-subunit. The G protein is functionally located between membrane receptors whose structures are characterized by seven membrane-spanning domains and effectors that are enzymes responsible for the generation of intracellular second messengers or ionic channels, thereby playing its essential role as a molecular switch for intracellular signal initiation. The switch turns on when GTP binds, in exchange for prebound GDP, to the α-subunit (Gα), whereas it turns off upon the GTP hydrolysis due to the Gα a GTPase activity. The βγ-component plays a supporting role for the molecular switching and is also involved in signal transduction to certain effectors. One of the most exciting subjects to be currently studied as to the physiological roles of G proteins will be the mechanism by which the G protein-mediated second messenger system interacts (crosstalks) with the tyrosine kinase-mediated signaling system arising from other types of growth factor receptors.
In mammals, G-protein α, β, γ polypeptides are encoded by at least 16, 4 and 7 genes, respectively. Gα-subunits bind and hydrolyze GTP and have the sites for bacterial toxin-catalyzed ADP-ribosylation. A structural model of Gα-subunits can be defined on the basis of similarities between Gα and other members of the GTP-binding proteins. The resulting Gα model specifies the spatial relationship among the guanine nucleotide binding site, the binding site of the Gβγ-subunit complex, likely regions of effector and receptor interaction, and sites of cholera or pertussis toxin-induced modification. The architecture of the Gα core is the same as that of p21ras. Experimental evidence from immunological, molecular genetic and biochemical studies support the Gα model. The Gα-subunits alone were previously thought to act on the effector enzymes; However, recent evidence indicates that the Gβγ-dimer also plays an important part in effector activation.
Guanine nucleotide-binding regulatory proteins (heterotrimeric G proteins) are composed of α-, β- and γ-subunits, and they mediate a variety of intracellular signal transductions by coupling activated membrane receptors with effector enzymes and channels. Activated receptors catalyze the exchange of GDP bound to the α-subunits for cytosolic GTP, and GTP-bound α-subunits in turn regulate activities or functions of the effectors. The βγ-complex is not dissociable under physiological conditions, and it is indispensable for the GDP/GTP exchange reaction on the α-subunit. Recently, three kinds of lipid modifications have been found in the α- and γ-subunits. The first is the attachment of fatty acids, myristate (C14:0) or structurally related fatty acids to the N-terminal glycine residues of some members of the α-subunits. Another type of fatty acylation to be characterized is the linkage of palmitate (C16:0) to a number of α-subunits via a thioester bond at their cysteine residues. The third type of modification is polyisoprenylation (farnesylation or geranylgeranylation) and α-carboxyl methylation at the C-terminal cysteine residue of the γ-subunit. These modifications on the two subunits have been shown to play a critical role in not only protein-membrane interaction but also proper protein-protein interaction, both of which are required for the G protein function.
Receptors located on the cell surface are responsible for recognition of extracellular first messengers. The largest number of receptors belongs to a G protein-coupled receptor superfamily. They showed common structural features characterized by seven transmembrane regions and three connecting extracellular and intracellular loops. Stimulation of a receptor by an agonist activates the coupled G protein. From in vitro mutagenesis analyses of β2-adrenergic, muscarinic acetylcholine and α1-adrenergic receptors, it was shown that the intracellular third loop and carboxyl terminus of the receptor molecule are involved in coupling with a G protein. One intriguing observation was that mutation at a single site of the intracellular third loop could induce the active state of receptors without agonist stimulation. Receptor heterogeneity generated from distinct genes or alternative splicing can be seen at the intracellular third loop and carboxyl terminus that is assumed to play an important role of coupling with G proteins. There are several examples that receptor isoforms arising from alternative splicing have their own counterparts of G proteins.
Adenylylcyclase is a membrane bound enzyme that catalyzes the conversion of ATP to cyclic AMP. Studies on the regulation of adenylylcyclase have been hampered by the small amount of this enzyme in the cell as well as by the instability of the catalytic activity. Cloning of multiple adenylylcyclase isoforms (types I though VIII) has indicated the presence of a large enzyme family, which is further subdivided into several smaller groups. Members within the same group share similar biochemical properties. The multiplicity of adenylylcyclase is made through at least three distinct mechanisms. First, each isoform is encoded by a distinct gene. Second, multiple isoforms are generated through possible alternative splicing from the same gene. Third, there is a mechanism to generate a half-molecule of adenylylcyclase via alternative polyadenylation. Overexpression of a distinct isoform in insect cells followed by purification has enabled researchers to examine the role of each specific isoform in vitro. The results have suggested that each isoform is regulated through distinct mechanisms. For example, type I adenylylcyclase is inhibited in the presence of βγ-subunits, while type II is stimulated. Other isoforms such as types V and VI are not affected. On the other hand, Giα may directly inhibit each adenylylcyclase isoform. Further characterization of adenylylcyclase would be feasible using those clones in the future.
Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate to inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol. IP3 induces the release of Ca2+ from intracellular stores, and diacylglycerol acts as the physiological activator of protein kinase C. Several distinct PI-PLC enzymes have been identified from various cells. Based on the primary sequences, PI-PLC isozymes are divided into three families: PLC-β, PLC-γ, and PLC-δ. Substantial evidence has strongly suggested that G proteins regulate PI-PLC in various cell-stimulation systems and that there might be two distinct pathways (pertussis toxin-sensitive and pertussis toxin-insensitive). Recently, it has become apparent that β-type PLC isoforms are activated by the heterotrimeric G protein subfamily Gq. Careful studies using in vitro and in vivo reconstitution systems have further suggested that the α-subunits of Gq/11/16 specifically regulate PLC-β1 and PLC-β3 and that the βγ-subunits of the Gi subfamily interact with PLC-β2, which are considered to be responsible for the pertussis toxin-insensitive and the pertussis toxin-sensitive pathways, respectively. In this paper, involvement of G proteins in the regulation of phospholipase A2 and phosphatidylcholine-specific PLC and PLD is also discussed.
Direct G protein regulation of ion channels is one of the major mechanisms of the functional regulation of excitable cells by hormones and neurotransmitters. Recent extensive studies indicate that a number of channels are directly regulated by G proteins. In this short review, we focus on the molecular mechanism underlying G protein (GK) regulation fo cardiac muscarinic K+ channels. This channel was reported to be activated in the inside-out configuration by both exogenously applied α- and βγ-subunits of pertussis toxin-sensitive G proteins. There has been a longstanding controversy as to which of the subunits physiologically mediate the effect of GK on this channel. We have established the notion that GK physiologically activates cardiac muscarinic K+ channels through its βγ-subunit but not its α-subunit.