e G-protein Coupled Receptor Kinase 2

G-Protein Coupled Receptor Kinase 2
Juliette Leclerc '27 and Jason Fakler '27

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Contents:

I. Introduction and Background

G-Protein Coupled Receptor Kinase 2 (GRK2) is an intracellular serine/threonine protein kinase that phosphorylates G-Protein coupled receptors (GPCRs). GRK2 specifically acts on β2 adrenergic receptors (β2ARs), which are part of the Class A rhodopsin-like receptor family, and are typically found in the cardiac and lung pathways, on the surface of cardiomyocytes as well as in bronchioles in the airways. β2ARs are receptors that act on catecholamines, which include neurotransmitters such as dopamine, epinephrine, and norepinephrine. Increasing the circulatory concentration of these neurotransmitters is a well characterized neurohormonal mechanism of the sympathetic nervous system that is activated in response to the failing heart (Xu. et al, 2020).

G-Protein Coupled Receptors are membrane-bound receptors which contain an extracellular N-terminus, an intracellular C-terminus, and seven transmembrane . They are connected to ion channels or enzymes through G proteins, which are heterotrimeric intracellular membrane proteins with subunits 𝛼, β, and 𝛾, that bind to the guanine nucleotide. The extracellular binding of a ligand to a GPCR induces the activation of its associated G-protein, which occurs through the G-protein 𝛼 domain exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). In the GPCR’s active state, the G protein 𝛼 subunit is bound to guanosine triphosphate; when G-protein uses its GTPase activity to undergo GTP hydrolysis, the GPCR becomes inactivated. Once the GPCR is ligand-bound, the G-proteins dissociate into the 𝛼 subunit and a β𝛾 dimer. The β𝛾 subunits, together with the membrane phospholipids, recruit inactive GRK2 to the agonist-bound GPCR, allosterically activating the kinase for receptor phosphorylation. Following phosphorylation, β arrestins associate with the receptor, which triggers the uncoupling of the GPCR from its G-proteins and the endocytosis of the receptor.

II. General Structure


GRK2 comprises several domains, including a pleckstrin homology domain, which is important for localization of GRK2 to the cell membrane, and does so by binding the phospholipid membrane and G-protein β and 𝛾 subunits. GRK2 also contains a regulator of G protein signaling homology domain, which serves to maintain the 𝛼C helix and small lobe of the kinase domain in a competent configuration that keeps the kinase domain in an inactive and open conformation, until it needs to interact with a GPCR (Penela, 2019). The domain contains the active site of GRK2, which binds to . The bilobular fold of the catalytic domain is made up of an N-terminal and a C-terminal on either side of the catalytic cleft. It has a connecting the small and large lobes, and a , or phosphate-binding loop. The of the small lobe stabilizes the catalytic cleft, and the active site tether (AST, not imaged) of the large lobe stabilizes nucleotide and phosphate binding sites.

III. Regulation and Inhibition


GRK2’s phosphorylation of a β2 adrenergic receptor (β2AR) signals the release and binding of β-arrestin, a regulatory protein, which triggers the endocytosis of the receptor, and sterically hinders the binding of catecholamines to β2AR. GRK2 specifically phosphorylates residues within the carboxyl-terminal , namely , , , and . This leads to rapid desensitization of the receptor, in which the receptor’s response to a stimulus, in this case a catecholamine, is limited. Once desensitized, the receptor is sequestered, meaning that it dissociates from the membrane and is endocytosed into the cell via clathrin-coated vesicles. The receptor can be resensitized through dephosphorylation, and dissociation of the bound catecholamine and of β-arrestin. Once resensitized, β2AR can reassociate with the membrane and continue working as before.

GRK2 is also regulated through its phosphorylation by other kinases, including Protein Kinase A (PKA) and Protein Kinase C (PKC), among several others. PKA enhances GRK2’s ability to bind to Gβ𝛾 and the GPCR by phosphorylating Ser 685 (not imaged). PKC’s enhances GRK2’s binding and catalytic kinase activity towards GPCRs.

Inhibition of GRK2 can occur in its ATP binding site, as found in a hit-to-lead study by Xu et. al. Through competition for the active site, molecules that can bind to the ATP binding site can inhibit GRK2, eliminating its downstream effects. Some of these inhibitors include molecules such as βARKct, Balanol, and Takeda 103A. It has been shown that inhibition of GRK2 leads to improved cardiac function as a result of increased β2AR density and activity.

IV. Physiological Importance


Heart failure affects 6.5 million adults in the United States. Heart failure is a condition in which the heart cannot effectively pump enough blood to meet the body’s needs, and is due to the heart's inability to produce a strong myocardial contraction. The upregulation of GRK2 is a key feature of heart failure, as it leads to receptor dysfunction and cardiomyocyte death (Xu. et al, 2020).

As stated above, GRK2 causes inhibition of β2AR through the signaling of β-arrestin. This is an important process since the β2AR signaling cascade is vital for calcium release, which allows contractility in smooth and cardiac muscles, and is essential for a beating heart. β2AR is a Gs bound receptor, which is a stimulatory pathway for cyclic adenosine monophosphate (cAMP). Once the 𝛼 subunit of Gs is activated by the exchange of GDP for GTP, the G protein dissociates from the receptor and the 𝛼β𝛾 subunits dissociate from each other, leaving the 𝛼 subunit to connect to membrane-bound adenylyl cyclase. The stimulation of adenylyl cyclase by GTP-bound Gs causes the formation of cAMP from ATP. cAMP then activates cAMP-dependent protein kinase A (PKA), which causes a release of calcium, and subsequently, a beating heart.

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