Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

G proteins
Stimulatory Gs protein and inhibitory Gi protein of the adenylate cyclase system

As is well known, adenylyl cyclase, which catalyzes The formation of cyclic adenosine monophosphate from ATP, determines the intracellular level of cAMP—a secondary messenger that, in turn, regulates a wide range of biochemical processes. The activity of this enzyme, which is vital for Cell physiology, depends on extracellular factors, notably adrenaline, which interacts with a specific receptor in The Cell membrane. The signal from the receptor to adenylyl cyclase itself is transmitted by the stimulatory G_s protein. The latter is a heterotrimer composed of three distinct subunits. In animals, the 45 or 52 kDa G_s-a subunit is the product of expression of a single Gene. The difference in the length of its peptide chain is due to Alternative Splicing, which varies across different cell types. This subunit binds GDP or GTP and catalyzes the Hydrolysis of the latter. The G_s–GTP complex is Water-soluble and capable of activating adenylyl cyclase in the absence of other subunits.

The mode of action of the G_i protein is believed to be as follows. The complex of the G_s-a subunit and GDP interacts with the ß- and y-subunit complex, forming the inactive protein form: a-GDP-ß-y. The replacement of GDP by GTP in this complex occurs slowly, but it is dramatically accelerated when the G_s protein interacts with a stimulatory receptor located in the membrane. As a result of the GDP-to-GTP exchange, the trimeric Structure dissociates, releasing the ß–y complex, in which the y-subunit presumably acts as an anchor that allows the protein to attach to the membrane.

The released a-subunit, containing bound GTP, represents the active form of the protein. It forms a complex with adenylyl cyclase, thereby activating the enzyme. The result is an increase in intracellular cAMP concentration in response to a relatively weak extracellular signal, such as hormone binding to a receptor. The duration of this signal is determined by the time required for the a-subunit to carry out intramolecular hydrolysis of the bound GTP to GDP, after which the G_s-a–GDP complex recombines with the ß–y complex, returning the protein to its inactive state.

This model is supported by the fact that non-hydrolyzable GTP analogues, upon binding to the a subunit, keep adenylyl cyclase in a permanently activated state. The G_s-a subunit can undergo ADP-ribosylation, catalyzed by cholera toxin, which involves the modification of a specific Arginine residue (Arg-207). This leads to a reduction in the GTPase activity of the protein, resulting in constitutive activation of Adenylyl Cyclase and dysregulation of metabolic processes.

Cell membranes also contain receptors that, in response to binding external effectors, transmit signals that inhibit rather than activate adenylyl cyclase. The action of such receptors is mediated by a specialized "inhibitory" G_i protein and apparently proceeds as follows. Following effector binding, the receptor interacts with the G_i protein, which is composed of a, ß, and y subunits. As in the case discussed above, this leads to the dissociation of the protein into an a-subunit—which remains associated with the receptor until intramolecular hydrolysis of GTP takes place—and a ß–y complex. The latter consists of a ß-subunit, identical to that in the stimulatory G_s protein, and a y-subunit that likewise Functions as a membrane anchor. The ß–y complex binds the a-subunit of the stimulatory aG_s protein to form an inactive ternary complex, aG_s–GDP–ß–y, preventing it from activating adenylyl cyclase. Thus, the inhibitory G_i protein acts by releasing the inhibitor of the stimulatory G_s protein—the ß–y complex—in response to effector binding. The duration for which this complex remains free from contact with the a-subunit depends on how rapidly the latter cleaves the bound GTP.

In Conclusion, we note the widespread occurrence of G Proteins as signal transducers with a built-in timer mechanism. Approximately 150 genes for such proteins have been discovered. For many of them, the function remains unknown, and it has not even been established whether they belong to the stimulatory G_s or inhibitory G_i type; consequently, they are classified as G₀ proteins. At the same time, the number of G proteins involved in amplifying weak external signals is growing. For example, a G protein involved in Olfaction has been identified.

Many protein Translation factors are G proteins. As already mentioned, the EF-Tu factor, consisting of 393 amino acid residues, contains a G domain (residues 1–199). When this protein forms a complex with the EF-Ts factor, the latter essentially opens up the EF-Tu molecule and "inserts" GTP into it, displacing GDP. The resulting Conformational Changes in the G domain (residues 1–199) are transmitted to two other domains that interact with aminoacyl-tRNA and the ribosome. Following the Formation of the EF-Tu–GTP–ribosome–aminoacyl-tRNA complex, GTP hydrolysis is accelerated at the catalytic center of the G domain. Obviously, the GTPase activity of the G domain largely depends on whether the EF-Tu factor is isolated or complexed with The Ribosome and aminoacyl-tRNA. In other words, There is a multifaceted relationship between the Fine Structure of the catalytic center of the G domain and the Ligand-binding surface patches of the EF-Tu protein, which is presumably mediated by relatively minor yet functionally critical conformational changes.

Following the hydrolysis of GTP to GDP, the affinity of the EF-Tu factor for the ribosome and aminoacyl-tRNA drops sharply. If, for any reason, GTP hydrolysis occurs too rapidly, the EF-Tu factor will fail to properly organize the aminoacyl-tRNA–ribosome complex, leading to a decrease in translation fidelity.

Thus, G proteins function as highly efficient regulators capable of amplifying external signals and determining their duration. They achieve this by utilizing highly sensitive conformational changes associated with GTP or GDP binding, as well as their intrinsic GTPase activity, with all these properties subject to significant modulation upon complex formation with other proteins.

Class="center">12.3. The Stimulatory G_s Protein and Inhibitory G_i Protein of the Adenylyl Cyclase System

As is well known, adenylyl cyclase, which catalyzes the formation of cyclic adenosine monophosphate from ATP, determines the intracellular level of cAMP—a secondary messenger that, in turn, regulates a wide range of biochemical processes. The activity of this enzyme, which is vital for cell physiology, depends on extracellular factors, notably adrenaline, which interacts with a specific receptor in the cell membrane. The signal from the receptor to adenylyl cyclase itself is transmitted by the stimulatory G_s protein. The latter is a heterotrimer composed of three distinct subunits. In animals, the 45 or 52 kDa G_s-a subunit is the product of expression of a single gene. The difference in the length of its peptide chain is due to alternative splicing, which varies across different cell types. This subunit binds GDP or GTP and catalyzes the hydrolysis of the latter. The G_s–GTP complex is water-soluble and capable of activating adenylyl cyclase in the absence of other subunits.

The mode of action of the G_i protein is believed to be as follows. The complex of the G_s-a subunit and GDP interacts with the ß- and y-subunit complex, forming the inactive protein form: a-GDP-ß-y. The replacement of GDP by GTP in this complex occurs slowly, but it is dramatically accelerated when the G_s protein interacts with a stimulatory receptor located in the membrane. As a result of the GDP-to-GTP exchange, the trimeric structure dissociates, releasing the ß–y complex, in which the y-subunit presumably acts as an anchor that allows the protein to attach to the membrane.

The released a-subunit, containing bound GTP, represents the active form of the protein. It forms a complex with adenylyl cyclase, thereby activating the enzyme. The result is an increase in intracellular cAMP concentration in response to a relatively weak extracellular signal, such as hormone binding to a receptor. The duration of this signal is determined by the time required for the a-subunit to carry out intramolecular hydrolysis of the bound GTP to GDP, after which the G_s-a–GDP complex recombines with the ß–y complex, returning the protein to its inactive state.

This model is supported by the fact that non-hydrolyzable GTP analogues, upon binding to the a subunit, keep adenylyl cyclase in a permanently activated state. The G_s-a subunit can undergo ADP-ribosylation, catalyzed by cholera toxin, which involves the modification of a specific arginine residue (Arg-207). This leads to a reduction in the GTPase activity of the protein, resulting in constitutive activation of adenylyl cyclase and dysregulation of metabolic processes.

Cell membranes also contain receptors that, in response to binding external effectors, transmit signals that inhibit rather than activate adenylyl cyclase. The action of such receptors is mediated by a specialized "inhibitory" G_i protein and apparently proceeds as follows. Following effector binding, the receptor interacts with the G_i protein, which is composed of a, ß, and y subunits. As in the case discussed above, this leads to the dissociation of the protein into an a-subunit—which remains associated with the receptor until intramolecular hydrolysis of GTP takes place—and a ß–y complex. The latter consists of a ß-subunit, identical to that in the stimulatory G_s protein, and a y-subunit that likewise functions as a membrane anchor. The ß–y complex binds the a-subunit of the stimulatory aG_s protein to form an inactive ternary complex, aG_s–GDP–ß–y, preventing it from activating adenylyl cyclase. Thus, the inhibitory G_i protein acts by releasing the inhibitor of the stimulatory G_s protein—the ß–y complex—in response to effector binding. The duration for which this complex remains free from contact with the a-subunit depends on how rapidly the latter cleaves the bound GTP.

In conclusion, we note the widespread occurrence of G proteins as signal transducers with a built-in timer mechanism. Approximately 150 genes for such proteins have been discovered. For many of them, the function remains unknown, and it has not even been established whether they belong to the stimulatory G_s or inhibitory G_i type; consequently, they are classified as G₀ proteins. At the same time, the number of G proteins involved in amplifying weak external signals is growing. For example, a G protein involved in olfaction has been identified.

Many protein translation factors are G proteins. As already mentioned, the EF-Tu factor, consisting of 393 amino acid residues, contains a G domain (residues 1–199). When this protein forms a complex with the EF-Ts factor, the latter essentially opens up the EF-Tu molecule and "inserts" GTP into it, displacing GDP. The resulting conformational Changes in the G domain (residues 1–199) are transmitted to two other domains that interact with aminoacyl-tRNA and the ribosome. Following the formation of the EF-Tu–GTP–ribosome–aminoacyl-tRNA complex, GTP hydrolysis is accelerated at the catalytic center of the G domain. Obviously, the GTPase activity of the G domain largely depends on whether the EF-Tu factor is isolated or complexed with the ribosome and aminoacyl-tRNA. In other words, there is a multifaceted relationship between the fine STRUCTURE OF THE catalytic center of the G domain and the ligand-binding surface patches of the EF-Tu protein, which is presumably mediated by relatively minor yet functionally critical conformational changes.

Following the hydrolysis of GTP to GDP, the affinity of the EF-Tu factor for the ribosome and aminoacyl-tRNA drops sharply. If, for any reason, GTP hydrolysis occurs too rapidly, the EF-Tu factor will fail to properly organize the aminoacyl-tRNA–ribosome complex, leading to a decrease in translation fidelity.

Thus, G proteins function as highly efficient regulators capable of amplifying external signals and determining their duration. They achieve this by utilizing highly sensitive conformational changes associated with GTP or GDP binding, as well as their intrinsic GTPase activity, with all these properties subject to significant modulation upon complex formation with other proteins.