UCSFWhen the ligand binds to its receptor, subsequent signaling mechanism must transfer the binding or occupation of receptor into transmembrane singling, which we call occupancy-response. Most transmembrane signaling is accomplished by a small number of different molecular mechanisms to transduce many different signals. These structures in the process of transmembrane signaling involve receptors on the cell surface and within in the cell, as well as enzymes and other components that generate, amplify, coordinate, and terminate postreceptor signaling by chemical second messengers in the cytoplasm.

Generally, five basic mechanisms of transmembrane signaling are well understood. Each uses a different strategy to circumvent the barrier posed by the lipid bilayer of the plasma membrane. Today here we will discuss about the G protein receptor, which is a transmembrane receptor protein that stimulates a GTP-binding signal transducer protein (G protein) that in turn modulates production of an intracellular second messenger.

Many extracellular ligands act by increasing the intracellular concentrations of second messengers such as cAMP (adenylyl cyclase as the effector element), calcium ion, or the phosphoinositides. In most cases, they use a transmembrane signaling system with three separate components. First, the extracellular ligand is selectively detected by a cell-surface receptor, which causes the change in conformation. Second, the change  in conformation of the receptor in turn triggers the activation of a G protein located on the cytoplasmic face of the plasma membrane. Finally, the activated G protein then changes the activity of an effector element, usually an enzyme or ion channel, and this activity change of the effector element changes the concentration of the intracellular second messenger. It is the intracellular second messenger that makes the cellular response.

Common intracellular second messengers include cAMP, calcium ion, and phosphoinositides. There are many examples of such receptors, such as β adrenoceptors, glucagon receptors, thyrotropin receptors, and certain subtypes of dopamine and serotonin receptors.

G Protein ReceptorsG protein-coupled receptors (GPCRs) use a molecular mechanism that involves binding and hydrolysis of GTP, like shown in the figure on the left. This mechanism allows the transduced signal to be amplified. Becasue once the G-GTP formed, the duration of the activity of effector element like adenylyl cyclase depends on the longevity of G-GTP complex rather than on the receptor’s binding for its ligand. For example, norepinephrine may encounter its membrane receptor for only a few milliseconds, but the product of G-GTP from its binding may persist for tens of seconds before becoming inactive, which certainly amplifies the transduced signal of binding between norepinephrine and its receptor. Also, the molecular mechanism helps to explain the spare receptor theory in pharmacodynamics (for detail please refer to http://www.tomhsiung.com/wordpress/2014/06/the-properties-of-drugs-receptor-rationale/).

Receptor Regulation

G protein-mediated responses to drugs and hormonal agonists often attenuate with time. After reaching an initial high level, the response diminishes over seconds or minutes, even in the continued presence of the agonist. This “desensitization” is often rapidly reversible; a second exposure to agonist, if provided a few mintues after termination of the first exposure, results in a response similar to the initial response.

The mechanism of “desensitization” include two ways: phosphorylation of GPCRs’ cytoplasmic loops (residues, as shown in the figure below), and the endocytosis of GPCRs from the plasma membrane. The agonist-induced change in conformation of the receptor causes it to bind, activate, and serve as a substrate for a family of specific receptor kinases, called G protein-coupled receptor kinases (GRKs). The activated GRK then phosphorylates serine residues in the receptor’s carboxyl terminal tail. The presence of phosphoserines increases the receptor’s affinity for binding a third protein, β-arrestin, and the binding of β-arrestin to cytoplasmic loops of the GPCR diminishes the receptor’s ability to interact with G protein, thereby reducing the agonist response.

Upon removal of agonist, GRK activation is terminated, the β-arrestin binding affinity  is reduced, and the desensitization process can be reversed by cellular phosphatases (the receptor must be engulfed into cytoplasm first, where it can touch the cellular phosphatases), which turn the phosphorylated serine residues back to what they were before.

On the other hand, the β-arrestin binding also accelerates endocytosis of receptors from the plasma membrane. However, this endocytosis is helpful since endocytosis of GPCRs promotes their dephosphorylation (need the termination of GRK activity and to be dissociated with β-arrestin first) by a receptor phosphatease that is present at high concentration on endosome membranes. Then, the dephosphorylated GPCRs can return to the plasma membrane.

Of note is that several GPCRs if persistently activated instead would be traffic to lysosomes after endocytosis and degraded. This process effectively attenuates (rather than restores) cellular responsiveness. So, depending on the particular receptor, duration of activation and the particular conformational change caused by a specific agent (e.g., etorphine and enkephalins cause rapid internalization of the receptor whereas morphine dose not cause MOR internalization), endocytosis can contribute to either rapid recovery or prolonged attenuation of cellualr responsiveness.Regulation of GPCRs

Well-Established Second Messengers

The second messengers are not the patent of G Protein-Coupled Receptors. Other receptor strategies such as receptor tyrosine kinases (e.g., receptor of ANP) also involve second messengers.

Cyclic Adensine Monophosphate (cAMP)

Acting as an intracellular second messenger, cAMP mediates such hormonal responses as the mobilization of stored energy (the breakdown of carbohydrates in liver or triglycerides in fat cells stimulated by β-adrenomimetic catecholamines), conservation of water by the kidney (mediated by vasopressin), Ca2+ homeostasis (regulated by parathyroid hormone), and increased rate and contractile force of heart muscle (β-adrenomimetic catecholamines). It also regulates the production of adrenal and sex steroids (in response to corticotropin or follicle-stimulating hormone), relaxation of smooth muscle, and many other endocrine and neural processes.

cAMP exerts most of its effects by stimulating cAMP-dependent protein kinases. These kinases are composed of a cAMP-binding regulatory (R) dimer and two catalytic (C) chains. When cAMP binds to the R dimer, active C chains are released to diffuse through through the cytoplasm and nucleus, where they transfer phosphate from ATP to appropriate substrate proteins, often enzymes. When the hormonal stimulus stops, the intracellular actions of cAMP are terminated by an elaborate series of enzymes. cAMP-stimulated phosphorylation of enzyme substrates is rapidly reversed by a diverse group of specific and nonspecific phosphateases. cAMP itself is degraded to 5′-AMP by several cyclic nucleotide phosphodiesterases (PDE).

Phosphoinositides and Calcium

Another well-studied second messenger system involves hormonal stimulation of phosphoinositide hydrolysis. Some of the hormones, neurotransmitters, and growth factors that trigger this pathway bind to receptors linked to G proteins, whereas others bind to receptor tyrosine kinases. In all cases, the crucial step is stimulation of a membrane enzyme, phospholipase C (PLC), which splits a minor phospholipid component of the plasma membrane, phosphatidylinositol-4,5-bisphosphate (PIP2), into two second messengers, diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3 or InsP3). Diacylglycerol is confined to the membrane, where it activates a phospholipid- and calcium-sensitive protein kinase called protein kinase C. IP3 is water-soluble and diffuses through the cytoplasm to trigger release of Ca2+ by binding to ligand-gated calcium channels in the limiting membranes of internal storage vesicles. Elevated cytoplasmic Ca2+ concentration resulting from IP3-promoted opening of these channels promotes the binding of Ca2+ to the calcium-binding protein calmodulin, which regulates activities of other enzymes, including calcium-dependent protein kinases. With its multiple second messengers and protein kinases, the phosphoinositide signaling pathway is much more complex than the cAMP pathway.

As in the cAMP system, multiple mechanisms damp or terminate signalling by this pathway. IP3 is inactivated by dephosphorylation. Diacylglycerol is either phosphorylated to yield phosphatidic acid, which is then converted back into phospholipids, or it is deacylated to yield arachidonic acid. Ca2+ is actively removed from the cytoplasm by Ca2+ pumps.

Cyclic Guanosine Monophosphate (cGMP)

Unlike cAMP, the ubiquitous and versatile carrier of diverse messages, cGMP has established signalling roles in only a few cell types. In intestinal mucosa and vascular smooth muscle, the cGMP-based signal transduction mechanism closely parallels the cAMP-mediated signalling mechanism. Ligands detected by cell-surface receptors stimulate membrane-bound guanylyl cyclase to produce cGMP, and cGMP acts by stimulating a cGMP-dependent protein kinase. The actions of cGMP in these cells are terminated by enzymatic degradation of the cyclic nucleotide and by dephosphorylation of kinase substrates.

Increased cGMP concentration causes relaxation of vascular smooth muscle by a kinase-mediated mechanism that results in dephospho rylation of myosin light chains. Examples include atrial natriuretic peptide (ANP), nitric oxide (NO) induced by vasodilator agents such as acetylcholine and histamine.