Question

Write out the GPCR pathway showing targets of the alpha subunit.

Answer 1

G Protein Coupled Receptor Pathway

• G proteins, also known as guanine nucleotide-binding proteins, involved in transmitting signals and function as molecular switches.
• G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, serpentine receptor, and G protein-linked receptors (GPLR),
• It constitute a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and ultimately, cellular responses.
• GPCRs are responsible for every aspect of human biology from vision, taste, sense of smell, sympathetic and parasympathetic nervous functions, metabolism, and immune regulation to reproduction.
• 45% of all pharmaceutical drugs are known to target GPCRs

Structure of GPCR can be divided into three parts:

1. 1.The extra-cellular region, consisting of the N terminus and three extracellular loops (ECL1–ECL3);
2. The TM region, consisting of seven a-helices(TM1– TM7)
3. The intracellular region, consisting of three intra- cellular loops (ICL1–ICL3), an intracellular amphipathic helix (H8), and the C terminus .

G protein complexes are Made up of

1. 23 alpha (α)
2. 7 beta (β)
3. 12 gamma (γ) subunits.

• Beta and gamma subunits can form a stable dimeric complex referred to as the beta-gamma complex
• The extracellular region modulates ligand access; the TM region forms the structural core, binds ligands and transduces this information to the intracellular region through conformational changes, and the intracellular region interfaces with cytosolic signalling proteins
• The α subunits fall into four families (Gs, Gi, Gq, and G12/13) which are responsible for coupling GPCRs to relatively distinct effectors.

MECHANISM

1. When an agonist binds to a GPCR, there is a conformational change in the receptor that is transmitted from the ligand-binding pocket to the second and third intracellular loops of the receptor which couple to the G protein heterotrimer.
2. GPCR results in a conformation change in the receptor that is transmitted to the bound Gα subunit .
3. This conformational change causes the α subunit to exchange its bound GDP for GTP.
4. Binding of GTP activates the α subunit and causes it to release both the βγ dimer and the receptor, and active signaling molecules, both the GTP-bound α subunit and the βγ heterodimer become and active signaling molecules .
5. The interaction of the agonist-bound GPCR with the G protein is transient; following activation of one G protein, the receptor is freed to interact with other G proteins.

Principal signal transduction pathways involving the G protein–coupled receptors:

1. cAMP signal pathway
2. the phosphatidylinositol signal pathway

Depending on the nature of the subunit, the active, GTP-bound form binds to and regulates effectors such as adenylyl cyclase (via Gs ) or phospholipase C (via Gq ).

The subunit can regulate many effectors including ion channels and enzymes such as PI3-K(phosphoinositide 3- kinase).

The G protein remains active until the GTP bound to the subunit is hydrolyzed to GDP.

The α subunit has a slow intrinsic rate of GTP hydrolysis that is modulated by a family of proteins termed regulators of G protein signaling (RGSs).

The RGS proteins greatly accelerate the hydrolysis of GTP and are potentially attractive drug targets.

Once the GDP bound to the α subunit is hydrolyzed to GDP, the βγ subunit and receptor recombine to form the inactive receptor-G protein heterotrimer basal complex that can be reactivated by another ligand-binding event

The main targets for G-proteins, through which GPCRs control different aspects of cell function are

1. adenylyl cyclase, the enzyme responsible for cAMP formation
2. phospholipase C, the enzyme responsible for inositol phosphate and diacylglycerol (DAG) formation
3. ion channels, particularly calcium and potassium channels
4. Rho A/Rho kinase, a system that controls the activity of many signalling pathways controlling cell growth and proliferation, smooth muscle contraction

The Adenylyl cyclase/cAMP signal pathway

1. cAMP is a nucleotide
2. Synthesized within the cell from ATP by membrane-bound, adenylyl cyclase
3. Produced continuously
4. Inactivated by hydrolysis to 5´-AMP, by the Phosphodiesterases
5. Involved in: Energy metabolism Cell division and cell differentiation Ion transport, ion channels Contractile proteins in smooth muscle

Nucleotide Exchange Factors (Gefs) -  Integrate extracellular signals from membrane receptors to produce cytoskeletal changes

• This pathway provides an additional effector system for cAMP signaling and drug action that can act independently or cooperatively with PKA
• Activation of diverse signaling pathways, regulate : Phagocytosis Progression through the cell cycle Cell adhesion Gene expression Apoptosis

Phosphodiesterases - Hydrolyze the cyclic 3',5'-phosphodiester bond in cAMP and cGMP

• Drug targets for : Asthma Cardiovascular diseases such as heart failure Atherosclerotic coronary and peripheral arterial disease Neurological disorders

The Phospholipase C/ inositol phosphate system

• PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ (PLCβ),
• Which splits it into DAG and inositol (1,4,5) trisphosphate (IP3)
• Both function as second messengers - IP3 receptor- a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum
• Diacylglycerol and protein kinase C
• DAG, unlike the inositol phosphates, is highly lipophilic and remains within the membrane
• Binds to a specific site on the PKC molecule, which migrates from the cytosol to the cell membrane in the presence of DAG, thereby becoming activated
• Kinases in general play a central role in signal transduction, and control many different aspects of cell function
• The Rho/Rho kinase system
• Activated by certain GPCRs (and also by non-GPCR mechanisms), which couple to G-proteins of the G12/13 type
• Rho-GDP, the resting form, is inactive
• When GDP-GTP exchange occurs, Rho is activated - In turn activates Rho kinase
• involved in Smooth muscle contraction and proliferation, angiogenesis and synaptic remodeling
• Important in the pathogenesis of pulmonary hypertension