Two Isoforms of G Protein-Coupled Receptor Kinase 4 Identified by Molecular Cloning

doi:10.1006/bbrc.1994.1306

Biochemical and Biophysical Research Communications

Volume 199, Issue 2, 15 March 1994, Pages 848-854

https://doi.org/10.1006/bbrc.1994.1306 Get rights and content

Abstract

Using PCR we found that G protein-coupled receptor kinase4 (GRK4) mRNA is expressed only in brain out of several tissues tested. In the brain two amplification products were generated. Sequence analysis revealed that the two fragments differed only by the presence or absence of an in-frame-sequence of 96 bp/32 aminoacids, located near the N-terminal of the kinase. This demonstrates the existence of two isoforms of GRK4 which were named GRK4A and GRK4B in the presence or absence of the insert, respectively. This is the first evidence that, within the GRKs gene family, different isoforms do exist.

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Cited by (30)

Proteomics analysis of G protein-coupled receptor kinase 4-inhibited cellular growth of HEK293 cells
2019, Journal of Proteomics
Citation Excerpt :
In mammals, the seven GRK members are known as GRK1-7, which have been named according to their order of discovery [2]. Based on their function and structure, GRKs are grouped into three subfamilies: the GRK1 subfamily (rhodopsin kinase subfamily), consisting of GRK1 and GRK7, which are specifically distributed in the retina of mammals with phosphorylated rhodopsin; the GRK2 subfamily (β-adrenergic receptor kinase subfamily), containing GRK2 and GRK3, which are diffusely distributed and regulate the β-adrenergic receptor maintaining extensive activity; and the GRK4 subfamily including GRK4, GRK5, and GRK6, among which GRK5 and GRK6 are widespread, similar to GRK2 and GRK3, and GRK4 expression is limited to a few tissues: testis, myometrium, kidney and brain [4–7]. In addition to phosphorylating hundreds of GPCRs, GRKs act as versatile regulators of non-GPCRs, such as receptor-tyrosine kinases (RTKs) and a variety of cytosolic or nuclear receptors via such pathways as MEK/MAPK, CAMP, PDGFR, IGF-IR, and ERK1/2 signaling [8–10].
G protein-coupled receptor kinases (GRKs) are involved in a wide range of cellular physiology and pathological activities by specifically phosphorylating activated G protein-coupled receptors (GPCRs) to terminate GPCR signaling, or through regulating non-GPCR substrates. We recently reported that overexpression of GRK4 halts cell proliferation and induces cellular senescent phenotype in HEK293 cells. In this study, a quantitative proteomic assay was performed to analyze the protein profiles between HEK293 cells expressing and not expressing GRK4. Results revealed 39 upregulated and 59 downregulated differently expressed proteins (DEPs) in a total of 4124 identified proteins. Gene ontology (GO) annotation and functional enrichment revealed that the DEPs were related to metabolic processes regulated by the binding of these RNA/proteins under the biological processes. The Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis showed pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression are involved in. Immunoblotting validation of expression of six key target proteins, CALM1, STAT3, CDK1, CDK6, TOP2A, and GRK4, which speculatively maintain abnormal activity in the above pathways, was consistent with the results of proteomics analysis. Lastly, a biological phenotype assay confirmed that GRK4 promoted HEK293 cell growth blockage and G1/0 arrest. Taken together, this study identified some novel molecules that involve in GRK4 signaling and provided valuable information for further studying the mechanisms underlying GRK4-induced proliferative inhibition.
A quantitative proteomic assay was performed in HEK293 cells expressing and not expressing GRK4 39 upregulated and 59 downregulated differently expressed proteins (DEPs)were identified.
DEPs involved in pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression.
Biological phenotype assay confirmed that GRK4 prompted HEK293 cell growth blockage and G1/0 arrest.
Receptors for neuronal or endocrine signalling molecules as potential targets for the control of insect pests
2014, Advances in Insect Physiology
Citation Excerpt :
It appears to interfere with the coupling between the receptor and G protein. In mammals, seven GRKs were found (as reviewed by Pitcher et al., 1998; Premont et al., 1995) and are divided in three subfamilies: (i) GRK1 (rhodopsin kinase) (Lorenz et al., 1991; Shichi and Somers, 1978) and GRK7 (cone opsin kinase) (Hisatomi et al., 1998); (ii) GRK2 and 3 (respectively β-adrenergic receptor kinase (βARK) 1 and 2) (Benovic et al., 1986, 1989, 1991); and (iii) GRK4 (Ambrose et al., 1992; Premont et al., 1994; Sallese et al., 1994), GRK5 (Kunapuli and Benovic, 1993) and GRK6 (Benovic and Gomez, 1993; Haribabu and Snyderman, 1993). All GRKs consist of three functional domains: an N-terminal regulator of G protein signalling homology domain, a central catalytic domain (serine/threonine protein kinase domain), and a C-terminal membrane targeting domain.
In metazoans, neuronal and endocrine communication is based on the release of extracellular signalling molecules that are recognised in a physiological concentration range by specific receptor proteins present in the target cells. These receptors will elicit a cellular response upon activation by their physiological agonist. A highly diverse repertoire of naturally occurring receptor agonists has already been discovered. Peptides, proteins and biogenic amines constitute the most diverse agonist classes. Most of these interact with G protein-coupled receptors (GPCRs), the largest category of signal transducing receptors that controls virtually every physiological process in metazoans. For more than two decades, insect GPCRs have been hailed for their potentially excellent aptitude to serve as pharmacological targets for the development of novel products for insect pest control. In this review, we will address this issue and enumerate reasons why it would be worth investing more in these targets.
Characterization of G protein-coupled receptor kinase 4 and measuring its constitutive activity In Vivo
2010, Methods in Enzymology
Citation Excerpt :
Shortly thereafter, the name GRK4 was given to the protein due to its similarity to the other GRK family members (Inglese et al., 1993). GRK4 exists as four splice variants: GRK4α is the full-length protein, GRK4β is missing exon 2 (32 amino acids) which contains the PIP2 binding region (Pitcher et al., 1996), GRK4γ is missing exon 15 (46 amino acids), and GRK4δ is missing both exons 2 and 15 (Premont et al., 1996; Sallese et al., 1994). Both exon 2 and 15 encode for the beginning and end of the RH domain, suggesting that the splice variants could have significantly different biological properties.
G protein-coupled receptor kinase 4 (GRK4) was originally identified in the brain and was initially thought to have a limited expression pattern and functionality; however, more recent studies have found that GRK4 is expressed in multiple tissues and cell types and that it contributes to cardiovascular disease. Additionally, human GRK4 exists as four splice variants and each variant can harbor at least three functionally relevant polymorphisms. The primary role of GRK4 is to phosphorylate G protein-coupled receptors (GPCR), which leads to desensitization of the G protein signaling mechanism while simultaneously recruiting β-arrestins and initializing the internalization of the receptor. Interestingly, GRK4 has been shown to be constitutively active in some, but not all, cases. A constitutive active GRK could lead to increased β-arrestin-mediated signaling while inhibiting traditional/canonical GPCR-mediated signaling mechanisms. Therefore, it is important to determine if GRK4 is constitutively active in a system. Measuring agonist-mediated activity of GRK4 is relatively straightforward since it inhibits second messenger signaling; however, only a few studies have directly examined the constitutive activity of GRK4 which requires techniques without an agonist. Since GRK4 has significant biological effects, identifying the mechanism underlying GRK4's constitutive activity and ligand-stimulated activity becomes increasingly important. Therefore, the methods provided here are designed to aid researchers in determining if GRK4 is expressed, and if so which GRK4 species is expressed, followed by procedures to identify if GRK4 is constitutively active in its model system. Last, procedures are explained for identifying if GRK4 is involved in its system in a nonconstitutive manner. The protocols described here are designed to be accessible to a wide range of scientists, which should allow for more laboratories to examine GRK4 constitutive activity as well as agonist-mediated activity.
G protein-coupled receptor kinase 4γ interacts with inactive Gα<inf>s</inf> and Gα<inf>13</inf>
2008, Biochemical and Biophysical Research Communications
Citation Excerpt :
Thus, the dual lysine to methionine mutation can be used to discriminate between kinase activity driven effects of GRK4γ and interactions with Gα subunit driven events. To examine Gαs-mediated signaling, we utilized WKY RPTCs; one of the few cells to express endogenous GRK4 [4–7]. WKY RPTCs were transfected with empty vector, GRK4γ, KD GRK4γ, or GRK2 and then stimulated with vehicle, or 30 nM or 10 μM Isoproterenol for 30 min in the presence of 500 μM IBMX, a phosphodiesterase inhibitor.
G protein-coupled receptors (GPCRs) are regulated by multiple families of kinases including GPCR kinases (GRKs). GRK4 is constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to hypertension. We examined, through co-immunoprecipitation, the interactions between GRK4γ and the Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gα_s-coupled GPCR signaling and lacks a PH domain, we hypothesized that GRK4γ would interact with active Gα_s, but not Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gα_s and Gβ to a greater extent than active Gα_s. GRK4γ also interacts with inactive Gα₁₃ and Gβ. Functional studies demonstrate that wild-type GRK4γ, but not kinase-dead GRK4γ, ablates isoproterenol-mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that binding to inactive Gα_s and Gβ may explain the constitutive activity of GRK4γ towards Gα_s-coupled receptors.
G protein coupled receptor kinases
2007, xPharm: The Comprehensive Pharmacology Reference
G protein-coupled receptor kinases (GRKs) belong to the AGC family of serine/threonine kinases. They phosphorylate agonist-activated G protein-coupled receptors (hence the name). This phosphorylation triggers the binding of arrestins to the receptors. Receptor:arrestin interaction then leads to the desensitization of G protein activation by the receptors and can also lead to internalization of the receptors and initiation of new signaling pathways.
Some G protein-coupled receptor kinases can also constitutively phosphorylate cytosolic proteins, including beta-tubulin …
Pathophysiological roles of G-protein-coupled receptor kinases
2005, Cellular Signalling
G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptors (GPCRs) to effect receptor phosphorylation and to initiate profound impairment of receptor signalling, or desensitization. GPCRs form the largest family of cell surface receptors known and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations. This review focuses on the physiological role of GRKs revealed by genetically modified animals but also develops the involvement of GRKs in human diseases as, Oguchi disease, heart failure, hypertension or rhumatoid arthritis. Furthermore, the regulation of GRK levels in opiate addiction, cancers, psychiatric diseases, cystic fibrosis and cardiac diseases is discussed. Both transgenic mice and human pathologies have demonstrated the importance of GRKs in the signalling pathways of rhodopsin, β-adrenergic and dopamine-1 receptors. The modulation of GRK activity in animal models of cardiac diseases can be effective to restore cardiac function in heart failure and opens a novel therapeutic strategy in diseases with GPCR dysregulation.

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Regular ArticleTwo Isoforms of G Protein-Coupled Receptor Kinase 4 Identified by Molecular Cloning

Abstract

Regular Article
Two Isoforms of G Protein-Coupled Receptor Kinase 4 Identified by Molecular Cloning