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GRKs

The superfamily of G protein-coupled receptors (GPCRs) contains upwards of 1000 distinct members that mediate the vast majority of responses to hormones, neurotransmitters, sensory stimuli, and various autocrine and paracrine factors. These integral membrane proteins have a conserved primary structure that contains seven hydrophobic regions that span the plasma membrane. Upon binding agonist, GPCRs undergo a conformational change that promotes coupling to heterotrimeric guanine nucleotide binding proteins (G proteins) that consist of α, β and γ subunits. The agonist-occupied GPCR acts as a guanine nucleotide exchange factor and activates the G protein by promoting the dissociation of GDP from the Gα subunit. The nucleotide-free Gα then binds GTP and the Gα-GTP and Gβγ subunits dissociate and interact with various effector proteins.

In order to ensure that extracellular stimuli are translated into intracellular signals of appropriate magnitude and specificity, GPCR signaling cascades are tightly regulated. GPCRs are subject to three principle modes of regulation: (i) desensitization, the process whereby a receptor becomes refractory to continued stimuli; (ii) internalization, where receptors are physically removed from the cell surface by endocytosis; and (iii) down-regulation, where total cellular receptor levels are decreased. GPCR desensitization is primarily mediated by second messenger dependent kinases, such as protein kinase A (PKA) and protein kinase C (PKC), and by GPCR kinases (GRKs). GRKs specifically phosphorylate activated GPCRs and initiate the recruitment of arrestins, which mediate receptor desensitization and internalization.

GRKs are found in metazoans and, in mammals, the seven GRKs can be divided into three subfamilies based on overall structural organization and homology: GRK1 (rhodopsin kinase) and GRK7; GRK2 (βARK1) and GRK3 (βARK2); and GRK4, GRK5 and GRK6. GRKs are serine/threonine kinases with a tripartite modular structure. A central ~330 amino acid catalytic domain is flanked by an ~180 residue N-terminal region that contains a regulator of G protein signaling (RGS) domain and an ~60-160 amino acid C-terminal lipid-binding domain. Interestingly, the X-ray crystal structure of GRK2 suggests that it can simultaneously bind to receptor, Gαq (through the RGS domain), and Gβγ (through a C-terminal pleckstrin homology domain), thus providing an effective mechanism to terminate signaling.

A critical component in modulating GRK function involves regulating the activity and cellular localization of GRKs. Phosphorylation appears to play an important role in regulating GRK activity. GRK2 phosphorylation by ERK1/2 inhibits GRK2 activity while phosphorylation by PKA, Src, and PKC results in increased activity. In contrast, GRK5 activity is attenuated by PKC phosphorylation but stimulated by autophosphorylation. GRK function is also regulated by interaction with a large number of additional proteins including G protein subunits, the GRK-interacting protein GIT1, caveolin-1, phosphoinositide 3-kinase, cytoskeletal proteins such as tubulin and actin, and various calcium binding proteins. Phospholipids also play an important role in regulating GRK function since Gβγ-mediated GRK2 activation is dependent on negatively charged phospholipids. Interestingly, while GRK4 subfamily members do not bind Gβγ subunits, these kinases share an amino-terminal lipid-binding domain that may facilitate receptor phosphorylation. These kinases also have the ability to associate with phospholipids via a carboxyl-terminal domain that is either palmitoylated (GRK4 and 6) or can directly bind phospholipids (GRK5). Thus, the immediate phospholipid environment may have a general and critical role in modulating GRK function.

While in vitro studies have provided important insight into GRK/receptor interaction, other studies have focused on manipulating GRKs in intact cell systems and model organisms. For example, transfection of antisense GRK constructs has revealed subtype-specific regulation of H2 histamine receptors (by GRK2), pituitary adenylate cyclase activating peptide (PACAP) type 1 and corticotrophin releasing factor (CRF1) receptors (by GRK3), metabotropic glutamate type 1 receptors (by GRK4), thyrotropin receptors (by GRK5), and calcitonin gene-related peptide (CGRP) receptors (by GRK6). Insight into GRK specificity/function has also been gained from transgenic mice where cardiac-specific overexpression of GRK2 or a carboxyl-terminal GRK2 mini-gene demonstrates specific in vivo effects on cardiac function. Functional knockouts of GRKs have also provided important physiological insight. Disruption of the mouse GRK2 gene results in embryonic lethality while a mutation that disrupts expression of Gprk2 (a Drosophila GRK4 homolog) leads to specific defects in embryogenesis. Disruption of the mouse GRK3 gene results in attenuated desensitization of olfactory and cholinergic responses while mutation of a GRK2/3 homolog in C. elegans results in defective chemosensation. A mouse GRK5 knockout leads to muscarinic supersensitivity and impaired receptor desensitization, while disruption of the GRK6 gene results in supersensitivity to the locomoter-stimulating effects of cocaine and amphetamine. These findings suggest that GRKs are involved not only in regulating signaling but may also have critical roles in regulating growth and development.

 

The Table below contains accepted modulators and additional information.

 

Family Members GRK1 GRK2 GRK3 GRK4
Other Names Rhodopsin kinase
G protein-coupled receptor kinase 1
βARK-1
G protein-coupled receptor kinase 2
βAPK2
G protein-coupled receptor kinase 3
IT11
G protein-coupled receptor kinase 4
Molecular Weight
(kDa)
67 kDa 80 kDa 80 kDa 67 kDa
Structural Data 563 aa 689 aa 688 aa 578 aa
Isoforms 2 C-terminal splice variants Not Known Not Known 4 splice variants
Species Mammals Mammals
Drosophila (1 GRK2/3 homolog)
C. elegans (1 GRK2/3 homolog)
Mammals
Drosophila (1 GRK2/3 homolog)
C. elegans (1 GRK2/3 homolog)
Mammals
Drosophila (1 GRK4/5/6 homolog)
C. elegans (1 GRK4/5/6 homolog)
Domain
Organization
RGS/protein kinase domains RGS/protein kinase/PH domains RGS/protein kinase/PH domains RGS/protein kinase domains
Phosphorylation
Sites
Autophosphorylation (activation)
PKA phosphorylation (inhibition)
Src phosphorylation (activation)
PKA phosphorylation (activation)
PKC phosphorylation (activation)
ERK1/2 phosphorylation (inhibition)
Not Known Not Known
Tissue
Distribution
Retina (rods and cones) Ubiquitous
Brain
Spleen
Skeletal muscle
Kidney
Lung
Ubiquitous
Brain
Testis
Olfactory tissue
Lung
Testis
Brain
Kidney
Myometrium
Subcellular
Localization
Rod and cone outer segments Cytoplasmic Cytoplasmic Not Known
Binding Partners/
Associated Proteins
Rhodopsin
Color opsins
Recoverin
G protein-coupled receptors
Gβγ subunits
Gαq/11
Calmodulin (P1431)
Tubulin
Caveolin-1
GIT1
PI 3-kinase
Actin (A3653, A2522)
Clathrin
Hsp90 (SRP4858)
Raf kinase inhibitor protein
G protein-coupled receptors
Gβγ subunits
Gαq/11
Calmodulin (P1431)
G protein-coupled receptors
Calmodulin (P1431)
Upstream
Activators
Rhodopsin G protein-coupled receptors
Gβγ subunits
Acidic phospholipids
G protein-coupled receptors
Gβγ subunits
Acidic phospholipids
G protein-coupled receptors
Downstream
Activation
Autophosphorylation PKA (P6998 (h), P2645 (b))
PKC (P3287 (h))
Src (S5439 (h)) phosphorylation
Not Known Not Known
Activators Isoprenylation
Rhodopsin
Gβγ subunits
PIP2 (P9763)
Acidic phospholipids
G protein-coupled receptors
PKC
PKA
Src
Gβγ subunits
PIP2 (P9763)
Acidic phospholipids
Palmitoylation
Inhibitors Recoverin
NCS-1
Calmodulin (P1431)
ERK1/2 (M3172)
α-actinin
Caveolin-1
Raf kinase inhibitor protein
Not Known Calmodulin (P1431)
PKC
Selective
Activators
Not Known Not Known Not Known Not Known
Physiological
Function
Rhodopsin deactivation β-Adrenoceptor desensitization Olfactory desensitization D1 dopamine responsiveness
Disease
Relevance
Oguchi disease
ESCS
Heart failure
Hypertension
Not Known Essential hypertension

 

 

Family Members GRK5 GRK6 GRK7
Other Names G protein-coupled receptor kinase 5 G protein-coupled receptor kinase 6 G protein-coupled receptor kinase 7
Molecular Weight
(kDa)
66 kDa 68 kDa 64 kDa
Structural Data 590 aa 576 aa 553 aa
Isoforms Not Known 3 C terminal splice variants Not Known
Species Mammals
Drosophila (1 GRK4/5/6 homolog)
C. elegans (1 GRK4/5/6 homolog)
Mammals
Drosophila (1 GRK4/5/6 homolog)
C. elegans (1 GRK4/5/6 homolog)
Most mammals (not in mouse)
Domain
Organization
RGS/protein kinase domains RGS/protein kinase domains RGS/protein kinase domain
Phosphorylation
Sites
Autophosphorylation (activation)
PKC phosphorylation (inhibition)
Calmodulin-dependent autophosphorylation (inhibition)  
Not Known Autophosphorylation
PKA phosphorylation (inhibition)
Tissue
Distribution
Ubiquitous
Lung
Kdney
Heart
Ubiquitous
Spleen
Lung
Liver
Skeletal muscle
Brain
Retina (cones)
Subcellular
Localization
Plasma membrane
Nucleus
Plasma membrane
Nucleus
Outer segments
Binding Partners/
Associated Proteins
G protein-coupled receptors
Calmodulin (P1431)
Actin (A3653, A2522)
α-actinin
Hsp90 (SRP4858)
G protein-coupled receptors
Calmodulin (P1431)
Na+/H+ exchanger regulatory factor
Hsp90 (SRP4858)
Color opsins
Upstream
Activators
G protein-coupled receptors G protein-coupled receptors Activated opsins
Downstream
Activation
Autophosphorylation Not Known Not Known
Activators Phospholipids
Autophosphorylation
Palmitoylation Isoprenylation
Inhibitors Calmodulin (P1431)
Ro 32-0432 (R137)
Ro 31-8220 (R136)
α-actinin
Actin (A3653, A2522)
PKC
Calmodulin (P1431)
α-actinin
PKA phosphorylation
Selective
Activators
Not Known Not Known Not Known
Physiological
Function
Cholinergic responsiveness D2 dopamine responsiveness
T cell & neutrophil chemotaxis
Cone deactivation
Disease
Relevance
Not Known Not Known ESCS

 

Abbreviations

βARK-1: β-Adrenergic receptor kinase-1
βARK-2: β-Adrenergic receptor kinase-2
mGluR1: Metabotropic glutamate receptor type 1
NCS-1: Neuronal calcium sensor
ND: Not determined
PIP2: Phosphatidylinositol 4,5-bisphosphate
Ro 32-0432: 2-{1-[3-(Amidinothio)propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl)-maleimide methanesulfonate
Ro 31-8220: 2-(8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide hydrochloride
ESCS: Enhanced S cone syndrome

 

References