Biochemistry, G Protein Coupled Receptors


Introduction

G protein-coupled receptors (GPCRs) are integral membrane proteins containing an extracellular amino terminus, seven transmembrane α-helical domains, and an intracellular carboxy terminus. GPCRs recognize a wide variety of signals ranging from photons to ions, proteins, neurotransmitters, and hormones. The human genome encodes nearly 800 GPCRs, representing over 3% of human genes. The GPCR superfamily comprises at least five structurally distinct subfamilies: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin receptor families. About 90% of all GPCRs belong to the rhodopsin family. [1] 

Impaired ligand concentration, GPCR protein expression, or mutation and signaling are implicated in many pathophysiological conditions, including central nervous system (CNS) disorders, cardiovascular and metabolic diseases, respiratory malfunctions, gastrointestinal disorders, immune diseases, cancer, musculoskeletal pathologies, and eye diseases. Targeting of GPCRs is hence widely utilized for therapeutic intervention; GPCRs correspond to 30% of all identified drug targets and remain major targets for new drug development.[2][3] Signal transduction through G proteins is the most prominent feature of GPCRs, initiated by a ligand-GPCR interaction at the cell surface level.

Fundamentals

Signaling by GPCRs

G protein-coupled receptors (GPCRs) are integral membrane proteins that form the fourth largest superfamily in the human genome. GPCRs were named for their common ability to associate with heterotrimeric G proteins (Gαβγ). The binding of extracellular ligands initiates the signal transduction cascade by triggering conformational changes in the receptor that promote heterotrimeric GTP-binding protein (G protein) activation. The G protein is associated with the plasma membrane at the cytoplasmic side, connecting the GPCR to either enzymes or ion channels. In some cases, G proteins interact with the GPCR before the receptor is activated; in other instances, G protein interacts with GPCR only after stimulation with a ligand. G proteins have three subunits (α,β, and γ). 

When it is inactive, the α subunit of the G protein is bound to guanosine diphosphate (GDP). However, when a GPCR is activated, it induces the α subunit to release GDP and instead binds to guanosine triphosphate (GTP). By doing so, GPCR acts like a guanine nucleotide exchange factor (GEF). The exchange of GDP with GTP results in a conformational change in the G protein, which leads to its activation. The α subunit of G protein has a GTPase activity, and once it hydrolyzes GTP to GDP, it becomes inactive. The GTPase activity of the α subunit of G protein is significantly enhanced when it interacts with a specific regulator of G protein signaling (RGS). RGS proteins function as subunit-specific GTPase activating proteins (GAPs). There are currently 25 known GAPs in the human genome.[4][5]

GPCRs activate various intracellular signaling, including generating second messengers such as cyclic AMP and inositol phospholipids. GPCRs that stimulate the production of cyclic AMP are often coupled to the stimulatory G protein (Gs), which activates adenylyl cyclase and increases cyclic AMP levels. However, binding an inhibitory G protein (Gi) to a GPCR can inhibit cyclic AMP synthesis. Both Gs and Gi are targets of bacterial toxins. Cholera toxin interferes with the activity of the α subunit by ADP-ribosylation, causing it to remain active. Sustained and elevated concentration of cycle AMP in intestinal epithelial cells results in diarrhea. Pertussis toxin that causes pertussis (whooping cough) also can hydrolyze ADP-ribosylation of the α subunit of Gi, which inhibits its binding with GPCR, which results in the α subunit to remain in the GDP-bound state and makes it unable to regulate downstream signaling events.[6][7]

In mammalian cells, GPCR-induced cyclic AMP results in the activation of the cyclic AMP-dependent protein kinase (PKA). Once activated, PKA can phosphorylate many proteins on serine/threonine sites. In the inactive state, PKA is composed of a complex of two catalytic subunits and two regulatory subunits. The binding of cyclic AMP to the regulatory subunits induces a specific conformational change and results in the dissociation of the complex, which leads to the activation of the catalytic subunits.

The regulatory subunits of PKA (also known as A-kinase) are important for the sub-cellular localization of PKA, which is facilitated by the interaction of A-kinase anchoring proteins (AKAPs) with the regulatory subunits. One of the well-known targets of PKA is CRE-binding protein (CREB); PKA phosphorylates CREB on a specific serine residue. Phosphorylation of CREB allows the recruitment of a transcriptional co-activator CREB-binding protein (CBP), which stimulates the transcription of various target genes.[8][9]   

Many GPCRs elicit their physiological function by activating an inositol phospholipid signaling pathway via phospholipase C-β (PLCβ). This particular pathway is mediated by a sub-class of GPCRs often coupled to a G protein q (Gq) that leads to the activation of PLCβ. Activated PLCβ can hydrolyze phosphoinositol bisphosphate (PIP2) to form 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).

While IP3 is known to interact with the IP3 receptors at the endoplasmic reticulum (ER) and results in intracellular calcium release, DAG is best known for its role in the activation of protein kinase C (PKC). In some other cases, GPCRs can directly affect the ion channel activity in the plasma membrane, thereby regulating the ion permeability and membrane potential of the membrane. Yet, other GPCRs can regulate ion channels indirectly by regulating the phosphorylation of signaling proteins such as PKA and PKC.[10]

Issues of Concern

GPCRs are the largest class of membrane-bound receptors and one of the most prevalent gene families. These receptors are activated by various stimuli such as hormones, neurotransmitters, chemokines, odorants, and others. GPCRs influence broad physiological processes such as neurotransmission, cellular metabolism, secretion, cell growth, and immune responses. The availability of the structure of certain GPCRs has provided a basis for developing GPCR-based therapeutics.

However, detailed structural knowledge of most GPCRs is still lacking, which presents a significant challenge to target GPCRs for new therapeutics. GPCRs are widely expressed in human cells and tissues and cross-talk with other signaling pathways, such as receptor tyrosine kinases (RTKs) and ion channels. As a result, often, the vast majority of drugs targeting GPCRs have unintended side effects. Finally, there are still many orphan GPCRs whose endogenous ligands and functions are not known.

Function

As GPCRs are the most common surface receptors, it is understandable that they are involved in a wide variety of physiological roles. GPCRs are involved in sight, taste, smell, behavior, mood, and immune system regulation. Even though the signaling molecules, types of GPCR, and mechanisms of action may differ for all these roles, they all involve certain extracellular signals that are converted into a cellular response.

For example, mood regulation is affected by GPCRs because these receptors in the mammalian brain can bind to different neurotransmitters and create different physiological responses. Some molecules that can bind to GPCRs in the brain and lead to different moods are dopamine, serotonin, and GABA.[11]

Mechanism

As discussed above, upon interaction with ligands or light activation, GPCRs transduce the signal into the cell by activating a cascade of biochemical events, which is initiated by catalyzing GDP-GTP exchange on heterotrimeric G proteins. Attenuation of GPCR signals in the presence of agonist/ligand is established by a coordinated series of events that are generally considered as three distinct events: receptor desensitization, sequestration/internalization, and downregulation. Desensitization begins within seconds of agonist exposure and is initiated by phosphorylation of the receptor.

Second messenger-activated protein kinases, including PKA and PKC, phosphorylate serine and threonine residues within the cytoplasmic loops and C-terminal tail domain of GPCRs. Phosphorylation of these sites, in turn, impairs the receptor–G protein binding efficiency and, hence signaling. Desensitization is not associated with any detectable change in the number of receptors present in cells and is rapidly reversed following the removal of agonists.[12][13][14]

Internalization of GPCRs (endocytosis/receptor sequestration) occurs more slowly than desensitization, taking place over the course of several minutes after agonist exposure. Most, but not all, GPCRs undergo sequestration in a dynamin-dependent manner. Dynamin is a large GTPase necessary for the fission of clathrin-coated vesicles from the plasma membrane. Moreover, GRK-mediated GPCR phosphorylation and binding of β-arrestin to the receptor are known to facilitate the clathrin-dependent endocytosis of many GPCRs. Re-sensitization of a sequestered GPCR requires that β-arrestin is dissociated, the receptor is dephosphorylated, and the bound ligand is removed.[12]

Prolonged activation of GPCRs results in their removal from the plasma membrane, which is commonly known as downregulation and is established through endocytosis. Many GPCRs undergo ligand-induced endocytosis through clathrin-coated pits and are mediated by beta-arrestins. However, certain GPCRs differ significantly in their ability to undergo endocytosis via coated pits, and there is strong evidence for receptor-specific and cell-type-specific differences in the endocytosis of GPCRs.[12]

Pathophysiology

More than 30 human diseases and syndromes are potentially associated with the loss or gain of GPCRs caused by mutations. Mutational activation of GPCR (ie, constitutively activating mutations), such as mutations in an α1B-adrenergic receptor, specifically enhances receptor/G protein coupling in the absence of agonist ligands which could lead to cellular transformation and cancer.

Certain inherited diseases, such as autosomal dominant non-autoimmune hyperthyroidism, are caused by activating mutations in the TSH receptor gene. Similarly, a gain of function mutation in the LHR gene is known to cause a gonadotropin-releasing hormone (GnRH)-independent precocious puberty in boys, also called familial male-limited precocious puberty (FMPP) or testotoxicosis. Jansen's metaphyseal chondrodysplasia is a rare autosomal dominant form of short-limb dwarfism caused by activating mutations in the PTH/PTHrP receptor.[15]

Loss of function of GPCRs due to amino acid substitutions, truncations by nonsense or frameshift mutations, insertions, deletions, and rearrangements are also associated with many human diseases. For example, mutation of vasopressin V2 receptor (antidiuretic hormone receptor, AVPR2) and aquaporin 2 (AQP2) genes are linked to nephrogenic diabetes insipidus syndrome. Mutations in the Inactivating mutations in the rhodopsin (RHO) gene are the most common cause of retinitis pigmentosa (RP), which causes retinal dystrophy with early onset of night blindness and subsequent loss of the visual field.[16]

Clinical Significance

G-protein coupled receptors and G-proteins are prevalent throughout the human body and are involved in a wide range of physiological functions. Mutations in these proteins can lead to various diseases, including retinitis pigmentosa and cholera. Retinitis pigmentosa, caused by a mutation in a GPCR, is an eye disease in which the retina is damaged. People with this disease have blurred vision and/or difficulty seeing in low-light conditions. This eye disease is an inherited disorder, and there is no treatment. Wearing sunglasses can protect the vision that remains. There are over 800 different GPCRs in the human body. [17] 

Mutations in different GPCRs would cause other conditions. Along with retinitis pigmentosa, recent studies have shown that mutations in these critical surface receptors can play a role in hypothyroidism, hyperthyroidism, nephrogenic diabetes insipidus, and fertility issues.

The G-protein itself can also be affected and need not be genetic. Cholera is caused by a bacteria that multiplies within the human intestine and secretes a protein called cholera toxin. This toxin penetrates the cells that line the intestine and modifies the G protein. The α subunit, which stimulates adenylyl cyclase, is the subunit modified. This modification prevents GTP hydrolysis and locks the G-protein in the active state. The constant stimulation of adenylyl cyclase results in a prolonged and excessive outflow of chloride ions and water into the gut. This leads to severe diarrhea and dehydration. This can quickly lead to death, so water and ions should be replenished as fast as possible. Treatment consists of rehydration and antibiotics.[18][19]


Details

Author

Saba Rehman

Author

Nader Rahimi

Editor:

Manjari Dimri

Updated:

7/30/2023 12:57:40 PM

References


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