How drugs works: Molecular aspect. Objective/Learning outcome:  G-protein and role.  Targets for G-proteins.  Signal transduction via second-messengers.  Adenylate cyclase/cAMP system.  Phospholipase C/insitol phosphate system

G-proteins coupled receptors: They are called G-proteins because of their interaction with the guanine nucleotides GTP and GDP. G-proteins consists of three subunits,α,β,Y

Guanine nucleotides bind to α-subunit, which has enzymic activity, catalysing the conversion of GTP to GDP. The β- and -Y subunit remain together as a βY-complex.

All three subunits are anchored to the membrane through a fatty acid chain, coupled to the G-protein through a reaction known as Prenylation. G-protein is freely diffusible in the plain of membrane, and they interact with several different receptors and effectors in an essentially promiscuous fashion.

In a resting state, the G-protein exists as unattached αβY- trimer, with GDP occupying the site on the α- subunit. When a GPCR is occupied by an agonist molecule, a conformational change occurs, causing the bound GDP to dissociate and to be replaced with GTP ( GDP/GTP exchange),

Which in turn causes the dissociation of the G-protein trimer, releasing α- GTP and βY-subunits; these are the ‘ active’ forms of the G-protein, These active forms diffuse into the membrane and can associate with various enzymes and ion channels, causing activation or inactivation as the case may be.

The process is terminated when the hydrolysis of GTP to GDP occurs through the GTPase activity of the α- subunit. The resulting α-GDP then dissociates from the effector and reunites with the βY-subunits complex completing the cycle.

The mechanism results in,amplification because a single agonist-receptor complex can activate several G-protein molecules in turn, and each of these can remain associated with the effector enzyme for long enough to produce many molecules of product.

The product is often a second messenger, and further amplification occurs before the final cellular response is produce. The main class of G-protein are Gs, Gi, and Gp.

Targets for G-proteins: Adenylate cyclase: the enzyme responsible for cAMP formation Phospholipase C: the enzyme responsible for inostiol phosphate and diacylglycerol formation. Ion channels: particularly calcium and potassium channels.

The adenylate cyclase: cAMP is a nucleotide synthesized within the cell from ATP by the action of a membrane-bound enzyme, adenylate cyclase. cAMP is produced continuously and inactivated by hydrolysis to 5’-AMP through the action of a family of enzyme known as phosphodiesterases.

Many different drugs hormones and neurotransmitters act on GPCR and produce their effects by increasing or decreasing the catalytic activity of adenylate cyclase, thus raising or lowering the concentration of cAMP in within the cell.

cAMP regulates many aspects of cellular function including-: enzymes involved in energy metabolism; cell division and cell differentiation; ion channels; and contractile proteins in smooth muscle. The varied effects of cAMP are brought about by a common mechanism namely the activation of protein kinase by cAMP.

Protein kinase regulate the function of many different cellular proteins by catalyzing the phosphorylation of serine and threonine residues using ATP as a source of phosphate groups. Phosphorylation can either activate or inhibit target enzyme or ion channel.

Other examples of regulation by cAMP- dependent protein kinase includes; the increased activity of voltage-activated calcium channels in heart muscle cells; phosphorylation of these channels increase the amount of Ca 2+ entering the cell during the action potential and, thus increases the force of contraction of the heart.

The phospholipase C/ inositol phosphate system: The phosphoinositide system, an important intracellular second messenger system, was first discovered by Hokin and Hokin in the 1950’s,recondite interests centered on the mechanism of salt secretion by nasal glands of seabirds.

They found that secretion was accompanied by increased turnover of minor class of membrane phosphoplipids known as phosphoinositidies collectively known as PI.

Michell Berridge found that many hormones which produce an increase in free intracellular Ca 2+ concentration which includes for example ; muscarinic agonists and α- adrenoceptors agonists acting on smooth muscle and salivary glands and antidiuretic hormone (vasopressin) acting on liver cells also increase PI turnover.

Subsequently it was found that one particular member of the PI family, namely phosphatidylinositol 4,5- bisphosphate (pip 2 ), which has additional phosphate group attached to the inositol ring, plays a key role.

Pip 2 is the substrate for a membrane- bound enzyme, PLC β, which splits it into diacylglycerol (DAG) and inostiol 1,4,5- triphosphate (IP 3 ) both of which functions as second messengers.

Inositol phosphates and intracellular calcium(IP 3 ):  IP 3 water-soluble mediator  acts on specific IP 3 receptors ligand - gated calcium channel present on the membrane of the endoplasmic reticulum.  IP 3 is converted inside the cell to the 1,3,4,5-tetraphosphate,IP 4,by a specific kinase.

 IP 3 role is to control the release of Ca 2+ from the intracellular stores. Diacylglycerol and protein kinase C (DAG):  DAG is highly lipophilic and remains in the membrane.  The major effect of DAG is to activate a membrane-bound protein kinase, protein kinase C (PKC).

These PKC catalyses the phosphorylation of a variety of intracellular proteins. The various PKC isoforms,like tyrosine kinase act on many different functional proteins, such as ion channels, receptors, enzymes (including other kinases) and cytoskeletal proteins.

 Kinases in general, play a central role in signal transduction and control many different aspects of cell function.  The DAG-PKC link provides a mechanism whereby GPCRs can moblise this army of control freaks.

Ion channels as target for G-protein:  GPRCs can control ion channel function directly by mechanism that do not involve second messengers such as cAMP or IP 3.e.g.

 in cardiac muscle muscarinic acetylcholine receptors are known to enhance K + permeability and similar mechanism in neurons, where opiate analgesics reduce excitability by opening the potassium channels.

 These actions are produced by direct interaction between the G-protein subunit and the channels without the involvement of the second messengers.

END OF LECTURE 3 THANK YOU