Significance Of The Heterotrimeric Gproteins In Intracellular Transmission

As the name implies, these proteins are trimers, consisting of an a, p, and 7 subunit. They are bound to the inner membrane and the subunit can bind the guanine nucleotides, GTP and GDP. G-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. There are more than 100 receptors (not including odor receptors) that utilize G-proteins, and there are at least 20 members of the G-protein family, with each member having its characteristic a, p, and 7 subunits. While the subunit is different for each G-protein, the 8/7 pair can be the same. However, all of the G-proteins share a similar structure. In regard to the opioid receptor, specifically the G-proteins transmit the signal from the intracellular part of the receptor to the effector. Adenylyl cyclase (AC), which is an inner membrane-bound enzyme, regulates the production of the secondary messenger, adenylyl cyclase. Other effectors that are G-protein-dependent include additional enzymes, like cyclic GMP phosphodiesterase, and transmembrane ion channels (Figure II-24).


Figure 11-24. Transmembrane changes following binding of an opioid to the external part of the receptor. By internal activation of secondary messengers, the close ion-channel is activated resulting in an increase of K+-efflux

In its resting conformation, the G-protein consists of a complex of the three subunit chains and a GDP molecule bound to the alpha subunit. The alpha subunit is in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the G-protein at the alpha subunit. This results in an exchange of bound GDP for GTP, which is more abundant in the cell than GDP. GTP causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the P-7 pair. The alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. The alpha subunit is also a GTPase, so the signal transduction is regulated at this level by hydrolysis of GTP to GDP and inorganic phosphate. Such hydrolysis can occur spontaneously or upon interaction with a GTPase activating protein, "GAP". The GDP-alpha subunit complex then binds to the 8/7 complex to reform the original trimeric protein.

Since the stimulation of the external receptor can activate a number of G-proteins, signal amplification can occur. While this is a desired response in many instances, control at this level is needed to modulate it. G-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the GTP is hydrolyzed. The duration of production of secondary messenger, like cyclic AMP, is determined by the rate of hydrolysis. In this sense, the G-protein acts as a nano-timer.

Although there is controversy over the role of the 8/7 subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. If different receptors act on the same G-protein, or if different G-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. If the same receptor acts on many G-proteins, or if one G-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger.

Following binding the G-proteins activates the membrane-bound effector, adenylyl cyclase (AC). This enzyme catalyzes the synthesis of cyclic AMP resulting in the formation of ATP, cAMP and pyrophosphate.

Because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger" (Figure II-25). The reverse reaction, the formation of ATP from cAMP and pyrophosphate, is catalyzed by a specific phosphodiesterase. cAMP is involved in a number of physiologic processes. For the breakdown of glycogen, stimulation of the 8-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic AMP. The activity of cAMP-dependent protein kinase (cAPK) requires cAMP in order to phosphorylate Ser and Thr residues on other cellular proteins. Glycogen phosphorylase is activated by cAPK, making glucose-6-phosphate available for glycolysis.

Adenylyl cyclase activity is regulated at a number of levels, including modulation of GTPase activity of Ga, phosphodiesterase activity, and protein phosphatases. Inhibitory G proteins, Gi, are analogous to the stimulatory G proteins, Gs, except for the exchange of GTP by GDP by the a-subunit and the subsequent inhibitory action of Gia on adenylyl cyclase.

Figure II-25. Activation of the secondary messenger system following binding of a narcotic analgesic

Ugand t

Receptor *

O,- Protein adenylyl cyclase second messenger

Figure II-25. Activation of the secondary messenger system following binding of a narcotic analgesic

Most of the activities in cells are controlled by kinases and phosphatases. The intracellular, C-terminal domains of many receptors have tyrosine kinase activity. Such receptors are usually monomers in their unliganded states, and contain only a single transmembrane segment. Ligand binding to these receptors stimulates tyrosine kinase catalytic activity in the intracellular domain of the receptor (Figure II-26), and such intracellular protein phosphorylation events are now well established as a means of transmembrane signal transduction. Structurally, though, it is unlikely that the signal from bound receptor to the kinase domain is mediated by a conformational change, as there is only a single transmembrane segment. Rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor PTKs are activated. This dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate Tyr residues in the other's tyrosine kinase domain. Such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means.

All these changes are reversed with an overexpression of activation when an opioid is antagonized by a specific antagonist such as naloxone with activation of the excitatory NMDA-(N-methyl-D-aspartate) receptor, resulting in a rebound with an increase in transmission of stimuli (Figure II-27).

The next step in the signaling pathway involves activation of an inner membrane-bound monomeric G protein known as Ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. Ras, being a G protein, is activated when its bound GDP in the resting state is replaced by GTP. It, too, has GTPase activity, but the half-life is too slow

Figure II-26. Secondary intracellular phosphorylation of ion-channels by means of proteine kinase A (PKA) results in an increase of outflux of K+ and a reduction of influx of Ca++, inducing hyperpolarisation of the neuronal cell. As a consequence the cell no longer responds to incoming stimuli

to allow for effective regulation of a signal. Another GTPase activating protein, GAP, increases the rate of GTP hydrolysis by Ras. A "kinase cascade" ensues, involving Raf (a Ser/Thr kinase), MAP kinase (also known as MEK, which is both a Tyr kinase and Ser/Thr kinase, and a family of proteins known as MAPKs or ERKs.

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