G-PROTEINS

G-PROTEINS

 

1- The discovery of G- proteins.

Martin Rodbell and his colleagues found out that a transducer provided the link between the hormone receptor (the discriminator) and the amplifier. A transducer is a substance or device, such as a piezoelectric crystal, microphone, or photoelectric cell that converts input energy of one form into output energy of another.

The first clue that G protein would be important in mediating hormonal action was provided in the 1970s by reports that GTP was required for hormonal stimulation of intracellular cAMP production (1991).

Alfred G. Gilman and his collaborators used genetic and biochemical techniques to identify and purify the G protein. They used lymphoma cells that normally can be activated by a receptor to form cyclic adenosine monophosphate (cAMP). cAMP is a second messenger that is used by different hormones (2000). The studies largely responsible for the identification and characterization of the G protein thus relied on the variant of S49 lymphoma cells, termed [cyc.sup-]. The [cyc.sup.-] variant was so named because it could not be stimulated by the usual activators of adenylylcyclase ( 1991).

            A mutated lymphoma cell was found to contain a normal receptor and a normal cAMP-generating enzyme but was yet unable to respond because it lacked the transducer. This was a good system to assay purified G proteins. A G-protein could be isolated from normal brain tissue and inserted in the mutated cell, thereby restoring its function.       

Alfred Gilman and Martin Rodbell were awarded the Nobel Prize in Physiology or Medicine in 1994 for their discovery and research on G-proteins and the role of these proteins in signal transduction in cells.         

The importance of G proteins was first reported in the 1970s and it was soon discovered that G proteins not only stimulate some hormones, but that they inhibit others. To date, there have been nine G proteins identified.

 

2- What are G proteins and their structure?

G-proteins are heterotrimeric guanine nucleotide-binding proteins that participate in signal transduction in many tissues. They couple surface hormone – receptor binding to effector systems such as adenylate cyclase, phospholipases, and ion channels located on the intracellular surface of the plasma membrane (1992).

The larger heterotrimeric G proteins are another family of G proteins which couple cell surface receptors to catalytic units that catalyze the intracellular formation of second messengers or couple the receptors directly to ion channels (2001).

Heterotrimeric signal transducing guanine nucleotide binding proteins (G proteins) couple extracellular receptor proteins to intracellular effector enzymes and ion channels, serving as critical mediators of cellular responses to external stimuli. These proteins are a subset of a larger family of guanosine triphosphate (GTP)-binding proteins of differing molecular weight and subunit composition that share a common mechanism of GTP-binding and hydrolysis that regulates protein activity (Levine, 1996).

The G-protein consists of three components: an alpha (α) component that is the activator portion of the G-protein, and beta (β) and gamma (γ) components that attach the G-protein to the inside of the cell membrane adjacent to the receptor protein (2000).

The multiplicity of G protein subunits facilitates great combinatorial variability, which, in part, accounts for the ability of G proteins to interact with many different receptor and effector proteins. Over 100 G protein-coupled receptors have been identified. The receptors share a common serpentine structure consisting of 7 membrane-spanning domains, and detect extracellular signals as diverse as light, odorants, hormones, growth factors, and neurotransmitters. As already stated, G proteins regulate many second messenger systems, including enzymes such as adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase, as well as ion channels (1996).

Eight unique adenylyl cyclase molecules have been identified. Various combinations of these provide a large number of possible αβγ complexes. The α subunits and the by complex have actions independent of those on adenylyl cyclase (2000).

Small G proteins are involved in many cellular functions. Members of the Rab family of these proteins regulate the rate of vesicle traffic between the endoplasmic reticulum, the Golgi apparatus, lysosomes, endosomes, and the cell membrane (2001).

The α subunit is bound to GTP. When a ligand binds to a G-coupled receptor, this (guanosine diphosphate) GDP is exchanged for GTP and the α subunit separates from the combined β and γ subunits. The separated α subunit brings about many biologic effects. The β and γ subunits do not separate from each other, and βγ also achieves a variety of effectors.

The intrinsic GTPase activity of the α subunit then converts GTP to GDP, and this leads to reassociation of the α with the βγ subunit and termination of effector activation.

Heterotrimeric G proteins relay signals from over 1000 receptors, and their effectors in the cells include ion channels and enzymes. These are 16 α, 6 β, and 12 γ genes, so a large number of subunits are produced, and they can combine in various ways. They can be divided into five families, each with a relatively characteristic set of effectors. The families are Gs, Gi, Gt, Gq, and G13 (2001).

            Many G proteins are modified by having specific lipids attached to them, i.e., they are lapidated. Trimeric G proteins may be myristolated, palmitoylated, or prenylated. Small G proteins may be prenylated.   

G-proteins are also important mediators of hormonal inhibition of insulin secretion. G proteins are also integral to the proper functioning of the pancreas because of their role in catalyzing the function of many hormones that operate on pancreatic cells (1991)

 

3- How do they work? (Functions and mechanisms).

A common way to translate a signal to a biologic effect inside cells is by way of nucleotide regulatory proteins or G proteins that bind GTP. GTP is the guanosine analog of ATP. When the signal reaches a G protein, the protein exchanges GDP for GTP. The GTP-protein complex brings about the effect. The inherent GTPase activity of the protein then converts GTP to GDP, restoring the resting state. The GTPase activity is accelerated by a family RGS (regulators of G protein signaling) proteins that accelerate the formation of GDP (2001).

            It is now apparent that there is a large family of G proteins and that these are part of the superfamily of GTPases. The G protein family can be classified according to sequence homology into four subfamilies, as illustrated in the table below.

            Some forms of αi stimulate K+ channels and inhibit Ca2+ channels, and some αs molecules have the opposite effects. Members of the Gq family activate the phospholipase C group of enzymes.

βγ complexes have been associated with K+ channel stimulation and phospholipase C activation. G proteins are involved in many important biologic processes in addition to hormone action (2000). Some of these are listed in the table below.

Abundant experimental data have accumulated in the past 25 years that strongly support the concept that G proteins act as important modulators of pancreatic islet function. Early evidence for Gs as regulator of insulin secretion can be found in reports that β-adrenergic agonists stimulate β-cell adenylylcyclase, raise cAMP levels, and release insulin ( 1991).

More extensive data have also been reported supporting the concept that Gi is a physiologically important β-cell regulatory protein. The first reports that epinephrine inhibited glucose-induced insulin secretion appeared more than 25 years ago. The ability of epinephrine to inhibit insulin secretion was shown to be dependent on α-adrenergic activity, and direct evidence for α-adrenergic receptors in the islet has been reported (1991).

 

Classes and functions of G proteins

Class or Type

Stimulus

Effector

Effect

Gs

    αs

 

   αolf

 

Glucagon, β-adrenergics

 

Odorant

 

> Adenylyl cyclase

 

> Adenylyl cyclase

 

Gluconeogenesis Lipolysis, glycogenolysis

Olfaction

 Gi

    α11

    α12

   

    αo

  

    αt

 

Acetylcholine

α2-Adrenergics

M2 cholinergics

Opioids, endorphins

 

Light

 

< Adenylyl cyclase

> Potassium channels

 

> Potassium channels

< Calcium channels

> cGMP

phosphodiesterase

 

Slowed heart rate

 

 

Neuronal electrical activity

Vision

Gq

    αq

 

    α11

 

M1 cholinergics

α2-Adrenergics

α2-Adrenergics

 

 

> Phospholipase C-β1

> Phospholipase C-β2

 

 

> Muscle contraction

> Blood pressure

G12

    α12

 

?

 

?

 

?

 

 

4- Activation and inactivation of G proteins.

 

G-protein activity is regulated by the binding and hydrolysis of GTP by the α subunit. In the basal (nonstimulated) state, G proteins exist in the heterotrimeric form with GDP tightly bound to the α chain. Upon receptor activation, a conformational change occurs in the α chain that facilitates the exchange of bound GDP for GTP, with subsequent dissociation of the α-GTP chain from the βγ dimer and the receptor ( 1996).     

The free α-GTP chain is now able to interact with effector enzymes and ion channels to regulate their activity. In addition, free βγ dimers modulate the activity of at least some effectors and participate in receptor desensitization. The interaction of α-GTP with the effector molecule is terminated by the hydrolysis of GTP to GDP by an endogenous GTPase. With hydrolysis of GTP to GDP, the α-GDP chain reassociates with the βγ dimer and the heterotrimeric G protein is ready for another cycle of receptor activation (1996).

Binding of the hormones with the receptor allows coupling of the receptor to a G-protein. If the G-protein stimulates the adenylyl cyclase – cAMP system, it is called a Gs protein, denoting a stimulatory G-protein. Stimulation of adenylyl cyclase, a membrane-bound enzyme, by the Gs-protein then catalyzes the conversion of a small amount of cytoplasmic adenosine triphosphate (ATP) into cAMP inside the cell ( 2000).

This then activates cAMP-dependent protein kinase, which phosphorylates specific proteins in the cell, triggering biochemical reactions that ultimately lead to the cell’s response to the hormone.

Once cAMP is formed inside the cell, it usually activates a cascade of enzymes. That is, a first enzyme is activated, which then activates another enzyme, which activates a third, and so forth.

The importance of this mechanism is that only a few molecules of activated adenylyl cyclase immediately inside the cell membrane can cause many more molecules of the next enzyme to be activated, which can cause still more molecules of the third enzyme to be activated, and so forth. In this way, even the slightest amount of hormone acting on the cell surface can initiate a powerful cascading activating force for the entire cell.

If binding of the hormone to its receptors is coupled to an inhibitory G-protein (denoted Gi-protein), adenylyl cyclase will be inhibited, reducing the formation of cAMP and ultimately leading to an inhibitory action in the cell. Thus, depending on the coupling of the hormone receptor to an inhibitory or stimulatory G-protein, a hormone can either increase or decrease the concentration of cAMP and phosphorylation of key proteins inside the cell (2000).

In the postsynaptic neurons, there are several types of second messenger systems. One of the most prevailing types in neurons uses the G-proteins. A G protein is attached to the portion of the receptor protein that protrudes to the interior of the cell (2000).

On activation by a nerve impulse, the α portion of the G-protein separates from the β and γ portions and then is free to move within the cytoplasm of the cell. Inside the cytoplasm, the separated α component performs one or more multiple functions, depending on the specific characteristic of each type of neuron (2000).

            To summarize, the favored theory explaining activation of G proteins involves a critical exchange of GTP for GDP on the α-subunit catalyzed by the interaction of the heterotrimeric G protein with the hormone-receptor complex. After association of the hormone-receptor complex with the heterotrimeric G protein and the GTP-GDP exchange, the β and γ subunits dissociate from the heterotrimeric complex. This allows the dissociated α-subunit-GTP complex to enter an activated state. It is this activated species that couples to effector systems, e.g., adenylylcyclase, to regulate effector-system function. The activated α-subunit possesses intrinsic GTPase activity that can hydrolyze the bound GTP to GDP, which terminates activation of the α-subunit. Thus, closely integrated activation-autodeactivation of the G-protein α-subunit completes the cycle and allows the α-subunit-GDP complex to reassociate with β and γ subunits to reform the heterotrimer and return to the quiescent state.

 

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