G-protein isolated tissues. Different techniques such as:

G-protein coupled receptors (GPCRs) consist of seven transmembrane proteins connected by six loops that form the largest family of cell receptors in eukaryotes. The receptor is integrated in the plasma membrane. GPCR interact with G protein in the plasma membrane. GPCRs are heterotrimeric, they have three subunits: ? subunit, ? subunit and ? subunit. Theses subunits act as the key downstream signalling molecules of GPCRs. There are two signal transduction pathways involving GPCRs: the cAMP signal pathway and phosphatidylinositol signal pathway. This essay will discuss the mechanism activation of GPCRs.

The idea of drugs bind to receptors on cell surfaces came from Paul Ehrlich (1854-1915). The term ‘receptive substance’ originated by John Langley (1852-1925) and further developed by Sir Henly Dale. Early researchers further developed the concept of affinity and efficacy in the field of GPCR research and pharmacology. They also introduced the properties of agonists, partial agonists and antagonists from the measurement of functional responses in isolated tissues. Different techniques such as: homology modelling and ligand screening, utilizing structure- and fragment based protocols have been applied in the discovery of ligand binding to GPCRs. To determine the structures and functions of different GPCRs , homology-modeling technique was adopted from bovine rhodopsin (Hill 2009). The crucial role of GPCRs provides researchers a new scope for GPCR targeted drug discovery. As of 2017, 40% of pharmaceutical drugs target GPCR and the receptor continue to be a major focus in researches.

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GPCR respond to diverse array of sensory signal mediators, such as light, odour, taste, hormones and neurotransmitters. The external signal is a small molecule that binds to the receptor leading to a conformational change. Binding of different ligands stimulates different downstream responses. The ligands bind to GPCRs either competitively or allosterically. Ligands bound to competitive site are classified as agonists, antagonists and inverse agonists (Brogi et al 2014). Conformational changes in GPCRs leads to the binding and activation of heterotrimeric G protein. A core domain located in the seven transmembrane region is responsible for GPCRs activation. The conformational change of core domain affects the conformation of C2 and C3 intracellular loop which are directly linked to H3 and H6 that are the key sites for G protein activation (Bockaert 1999). The protein further activates a cascade of signalling events that results in a change in cell function.

G? subunit binds GTP/GDP, G? and G? subunits are linked to the plasma membrane. In the resting state, G? is attached to GDP and is complexed with G??. Binding of agonist releases the bound GDP in exchange for GTP, catalysed by guanine-nucleotide exchange factors (GEFs). These changes weakened the binding of G? with receptor and G?? dimer resulting in a free G?-GTP complex that binds to and activates an effector protein. The rate of GTP hydrolysis is slow, GTPase-activating protein enhanced the process with the result of returning the system to its resting state (de Opakua et al 2017).


There are four types of G protein (G?s, G?i/o, G?q/11, and G?12/13) which each gives out different cellular signalling events. There are two inhibitory G? subunits: G?t and G?z. The effector of both G?s and G?i/o protein is the cAMP-generating enzyme and adenylyl cyclase (AC). AC catalyse the conversion of ATP to cAMP, stimulated by G protein, G?s. Activation of AC increases the concentration of intracellular cAMP.

However, G?i/o protein is responsible for the inhibition of AC, reducing intracellular cAMP level. The prolong activation of G?i/o-coupled receptor causes a cellular adaptive tissue response that leads to heterologous sensitization of AC, stimulating cAMP signal transduction. The activity of various ion channels and protein kinase A (PKA) depend on the concentration of cytosolic cAMP. Therefore, cAMP is a secondary messenger and PKA is a secondary effector.

G?s subunits activate all isoforms of AC and catalyze ATP to cAMP. The effects of cAMP are mediated through PKA. cAMP binds the regulatory subunits of PKA which releases and activates the catalytic subunits. A catalytic subunit enters the nucleus and phosphorylates one of its multiple targets, the CREB transcription factor. Phosphorylated CREB binds cAMP response element or CRE sequences with the coactivator p300/CBP. It stimulates transcription of CRE regulated genes (Billington and Penn 2003). G?s play an essential role in heterologous sensitization of specific isoforms of AC. Persistent activation of the dopamine D2 receptor leads to enhanced potency of forskolin and relative efficacy of ?-adrenergic receptor agonist in C6 glioma cells. Chronic activation of adenosine A3 receptor results in the forskolin-stimulated sensitization of adenylyl cyclase in the presence of GTP in CHO cells. G?s-insensitive AC% mutants do not support dopamine D2 receptor-induced sensitization whereas G?s-insensitive mutants of AC1 are sensitized by the persistent activation of the dopamine D2 receptor. Forskolin interacts with all nine of the AC isoforms, further enhanced in the presence of Gs. The regulation of Gs-coupled AC proteins depends on the AC activity achieved in the presence of forskolin and GTP. Addition of manganese uncouples AC from G protein regulation due to blunt sensitization, reflects the activity of AC catalytic units independent of G protein function. http://molpharm.aspetjournals.org/content/52/4/632.full#sec-14


Heterologous sensitization is shown in G?i/o-coupled receptors. Activation of G?i/o-coupled receptors regulate the activity of adenylyl cyclase, such as PKA, PKC and Raf-1 kinase. Pertussis toxin is an inhibitor of all isoforms of G?i/o. It modifies a cysteine residue near the C terminus of G?i/o subunit. G?t and G?z are also G? inhibitors. Only G?t is sensitive to the inactivation of pertussis toxin. Expression of G?t weakens heterologous sensitization through µ and ?-opioid receptors, and G?z is dependent on the isoform of AC expressed in cells (Brust et al 2015). Pertussis toxin treatment and overexpression of G?z resulted in the heterologous sensitization through µ-opioid receptor (MOR) in cells expressing AC5 but not AC6. Opioid receptors are inhibitors in the activation of G?i/o protein. Acute activation of the µ-opioid reduced ?-opioid receptor (DOR) -mediated heterologous sensitization. Acute activation of DOR, ?2 adrenergic (?2AR), or nociception/orphanin FQ peptide receptors (NOPr) reduced MOR-mediated heterologous sensitization. In cell SH-SY5Y, MOR, DOR, ?2AR, NOPr receptors all compete for the inhibition of G protein. The rank order of AC inhibition by a maximum concentration of full agonists acting at G?i/o is MOR, DOR, NOPr, ?2AR, CB1, and 5-HT1A.The order was determined by relative receptor expression (Levitt et al 2010). All of these receptors share a common pool of AC but the agonist-mediated activity of each receptor determines the proportion of AC pool used. Compartmentalization prevents two proteins from sharing effector molecules, leading to signalling specificity. In NG108-15 cell, DOR do not share G?i/o with ?2AR, resulting in an additive response as each receptor activates its own pool of effectors. In N18TG2 cell, agonists to DOR and cannabinoid (CB1) receptors activates G?i/o in an additive manner. A high level concentration of receptors compete for a limiting pool of G?i/o, whereas at low receptor concentration, agonists for two receptor types activate G?i/o in an additive manner. Artificially reducing the number of G protein by using pertussis toxin did not increase competition of receptors. The results showed that during sensitization, G?i/o are still able to inhibit adenylyl cyclase and cAMP, and be activated by receptors (Brust et al 2015).

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