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PubMed1996

hPop1: an autoantigenic protein subunit shared by the human RNase P and RNase MRP ribonucleoproteins.

The eukaryotic endonucleases RNase P and RNase MRP require both RNA and protein subunits for function. Even though the human RNase P and MRP RNAs were previously characterized, the protein composition of the particles remains unknown. We have identified a human a Caenorhabditis elegans sequence showing homology to yPop1, a protein subunit of the yeast RNase P and MRP particles. A cDNA containing the complete coding sequence for the human protein, hPop1, was cloned. Sequence analysis identifies three novel sequence motifs, conserved between the human, C. elegans and yeast proteins. Affinity-purified anti-hPop1 antibodies recognize a single 115 kDa protein in HeLa cell nuclear extracts. Immunoprecipitations with different anti-hPop1 antibodies demonstrate an association of hPop1 with the vast majority of the RNase P and MRP RNAs in HeLa cell nuclear extracts. Additionally, anti-hPop1 immunoprecipitates possess RNase P enzymatic activity. These results establish hPop1 as the first identified RNase P and MRP protein subunit from humans. Anti-hPop1 antibodies generate a strong nucleolar and a weaker homogeneous nuclear staining in HeLa cells. A certain class of autoimmune patient serum precipitates in vitro-translated hPop1. hPop1 is therefore an autoantigen in patients suffering from connective tissue diseases.

Amino Acid Sequence↗
PubMed1994

Assessment of sulfhydryl group in individual rat lens protein subunits during galactose cataract development.

A specific reagent DACM [N-(7-Dimethylamino-4-methyl-3-coumarinyl)maleimide] is used to study the -SH groups in lens proteins of normal and galactose cataractous rats. DACM when reacts readily with -SH groups form strong fluorescent adducts. The two -dimensional electrophoresis with DACM pre-labeled proteins is a simple and sensitive method for detecting -SH groups of protein subunit. In the present study, based on IEF/SDS-PAGE electrophoretically characterized soluble crystallins, describes specific changes in -SH groups of protein subunit during the development of galactose cataract. The contents of -SH groups of crystallins are progressively decreased in cataractous (5+) lens, the reduction of -SH content in alpha- and beta- crystallin protein subunits of the normal and cataractous lens proteins is also noticeable.

Animals↗
PubMed2001

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis.

The intraneuronal aggregation of phosphorylated high-molecular-weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper-phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D-iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain-mediated proteolysis. The rate of calpain-mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.

Amino Acid Sequence↗
PubMed2001

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics.

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Adult↗
PubMed2000

Content change of neurofilament protein subunits in experimental brain diffuse axonal injury by lateral head rotation.

OBJECTIVE: To explore the content change of neurofilament (NF) protein subunits in the experimental brain diffuse axonal injury (DAI) by lateral head rotation. METHODS: Twenty-four Sprague Dawley (SD) rats were equally divided into three injury groups (2 h, 12 h, and 24 h post injury) and one control group. The models of DAI were made in the injury groups by lateral head rotation. Western blotting technique was used to measure the content of NF68 (a kind of NF protein subunit) in the brainstem tissues among all the injured and control rats. The NF68 immunohistochemical staining was used in another six SD rats in order to observe the morphological changes in DAI. RESULTS: The NF68 content in the brainstem tended to decrease at 2 h post injury, decreased significantly at 12 h and continued its decrease at 24 h. NF56 and NF52, as the breakdown products of NF68, had a tendency to increase at 2-12 h after the injury, and amounted to a significantly higher level at 24 h. Microscopically, there were a lot of swelling neuronal axons in the ventral part of the medullar oblongata at 2 h after the injury. Some axons were disconnected, and axonal retraction balls formed on their proximal end. CONCLUSIONS: There is an occurrence of phosphorolysis within the brainstem in DAI by lateral head rotation. These reactions cause the breakdown of NF68, which results in the decrease of NF68 in content. It suggests that the breakdown of neurofilament protein subunits is an important reason for structur al destroy of neurofilaments in DAI.

Journal Article↗
PubMed1998

RNA-dependent activation of primer RNA production by influenza virus polymerase: different regions of the same protein subunit constitute the two required RNA-binding sites.

The capped RNA primers required for the initiation of influenza virus mRNA synthesis are produced by the viral polymerase itself, which consists of three proteins PB1, PB2 and PA. Production of primers is activated only when the 5'- and 3'-terminal sequences of virion RNA (vRNA) bind sequentially to the polymerase, indicating that vRNA molecules function not only as templates for mRNA synthesis but also as essential cofactors which activate catalytic functions. Using thio U-substituted RNA and UV crosslinking, we demonstrate that the 5' and 3' sequences of vRNA bind to different amino acid sequences in the same protein subunit, the PB1 protein. Mutagenesis experiments proved that these two amino acid sequences constitute the functional RNA-binding sites. The 5' sequence of vRNA binds to an amino acid sequence centered around two arginine residues at positions 571 and 572, causing an allosteric alteration which activates two new functions of the polymerase complex. In addition to the PB2 protein subunit acquiring the ability to bind 5'-capped ends of RNAs, the PB1 protein itself acquires the ability to bind the 3' sequence of vRNA, via a ribonucleoprotein 1 (RNP1)-like motif, amino acids 249-256, which contains two phenylalanine residues required for binding. Binding to this site induces a second allosteric alteration which results in the activation of the endonuclease that produces the capped RNA primers needed for mRNA synthesis. Hence, the PB1 protein plays a central role in the catalytic activity of the viral polymerase, not only in the catalysis of RNA-chain elongation but also in the activation of the enzyme activities that produce capped RNA primers.

Arginine↗
PubMed1998

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy.

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of 'excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.

Adolescent↗
PubMed1993

Beta gamma subunits of guanine nucleotide-binding proteins and regulation of spontaneous receptor activity: thermodynamic model for the interaction between receptors and guanine nucleotide-binding protein subunits.

We used a thermodynamic model to examine the interactions between receptor, guanine nucleotide-binding protein (G protein), and their ligands. The model describes the interactions as multiple equilibria occurring between three distinct protein species (receptor, G alpha subunit, and G beta gamma complex) and two small ligands, i.e., agonist (which interacts with receptor) and guanine nucleotide (which binds to G alpha). The equilibrium distribution of free and complexed species is determined by the total concentration of the components, the affinities that govern the biomolecular reactions, and the allosteric interactions that ligands exert on each other when they are simultaneously bound to the same species. These allosteric factors are given in terms of free energy coupling. The model explains a number of experimental observations, as follows. (i) Both GTP and GDP can reduce agonist affinity, whereas the agonist enhances the net binding of GTP and diminishes that of GDP. (ii) G beta gamma is more effective in reducing agonist-independent than agonist-dependent receptor activity. (iii) Removal of guanine nucleotides increases the ratio between agonist-independent and -dependent activation of G protein. The model leads to a number of interesting predictions. (i) Not only G alpha but also G beta gamma has effects on hormone binding. (ii) As long as the distribution of protein species is [G beta gamma] > [G alpha] > [receptor] (as often observed in the cell membrane), small changes in the concentration of G beta gamma do not alter the overall response induced by agonist. (iii) Agonist activity examined at low concentrations of guanine nucleotide is inevitably different from that observed at high concentrations, typical of intact systems. (iv) Differences in potencies and maximal effects for various guanine nucleotide analogues may reflect differences in their coupling constants that are experimentally measurable. The present model suggests several experimentally testable hypotheses that could be important in elucidating the activation mechanism and regulatory flexibility of G protein-dependent transduction systems.

Allosteric Regulation↗
PubMed1993

Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands.

The heme protein subunit of sulfite reductase (SiR-HP; M(r) 64,000) from Escherichia coli as isolated contains the isobacteriochlorin siroheme exchange-coupled to a [4Fe-4S] cluster in the 2+ oxidation state. SiR-HP in the presence of a suitable electron donor can catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia. Paramagnetic 1H NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates sulfite and nitrite. As a model, the cyanide complex of purified siroheme was also prepared. The NMR spectrum of isolated ferric low-spin siroheme-CN is consistent with spin density being transferred into the a2u molecular orbital, an interaction which is symmetry-forbidden in porphyrins. The pattern of proton NMR shifts observed for isolated ferric low-spin siroheme-CN is very similar to those obtained for the protein-cyanide complex. NMR spectra of the cyanide complex of SiR-HP were obtained in all three accessible redox states. The pattern of hyperfine shifts observed for the one-electron and two-electron reduced cyanide complexes is typical of those seen for [4Fe-4S] clusters in the 2+ and 1+ oxidation states, respectively. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned for all complexes studied utilizing deuterium substitution. The cyanide-, sulfite-, and nitrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coupling exists between siroheme and cluster in solution. Data are also presented for the existence of a secondary anion binding site, the occupancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than that of ligand binding to heme.

Cyanides↗
PubMed2004

Inhibition of the expression of the human RNase P protein subunits Rpp21, Rpp25, Rpp29 by external guide sequences (EGSs) and siRNA.

External guide sequences (EGSs) and siRNAs were targeted individually to the mRNA of three of the protein subunits of human RNase P, Rpp21, Rpp25 and Rpp29. The production of each of the three targets was inhibited in every specific case. In addition, some of the remaining protein subunits were also inhibited by these specific EGSs and the siRNAs. These data, in general, confirm previous results on the inhibition of a sub-group of all the protein subunits with an EGS against Rpp38. The effect of EGSs is apparent in 24 hours after transfection but the effect of siRNAs, which is comparable to the EGS data in amounts of inhibition, takes at least 48 to 96 hours to become evident. No general understanding of the mechanism of action of the siRNAs, in terms of which portion of a target mRNA they bind to for function, was apparent from the design of those used here.

Base Sequence↗
PubMed1993

Checkerboard immunoblotting recognizes twenty epitopes among the B subunit proteins of the cholera enterotoxin family.

Checkerboard immunoblotting (CBIB) was used to analyze the reactions of a series of monoclonal antibodies with proteins of the cholera enterotoxin (CT) family, including heat labile enterotoxins (LTs) produced by diarrheagenic strains of Escherichia coli and genetically engineered chimeric proteins in which single amino acids of the CT-B subunit protein or human (H) LT-B subunit protein were substituted for corresponding residues in porcine (P) LT-B. The result indicated that there were at least twenty different patterns of reactivity suggesting that there are at least twenty recognizable epitopes among the proteins studied. An epitope which includes Ala46 appears to be particularly important. This epitope is common to CT and H-LT but not P-LT, and the epitope is not blocked by the Gm1 ganglioside. Human convalescent sera react with this epitope. CBIB is a versatile technique for epitope analysis.

Amino Acid Sequence↗
PubMed2003

Chronic prenatal ethanol exposure alters ionotropic glutamate receptor subunit protein levels in the adult guinea pig cerebral cortex.

BACKGROUND: The superfamily of glutamate-gated ion channels mediates fast excitatory synaptic transmission in the central nervous system and is composed of the NMDA, AMPA, and kainate receptors. Binding studies have shown that chronic prenatal and/or neonatal ethanol exposure produces persistent effects on the numbers of some of these channels. However, whether or not this chronic ethanol exposure produces long-lasting effects on the expression of specific ionotropic receptor subunits remains an open question. METHODS: Timed pregnant Dunkin-Hartley strain guinea pigs received oral administration of one of the following regimens between gestational days 2 and 67: (1) 4 g of ethanol per kilogram of maternal body weight per day with ad libitum access to pellet food and water (ethanol group), (2) isocaloric sucrose- and pair-feeding with ad libitum access to water (sucrose group), or (3) isovolumetric water with ad libitum access to food and water (water group). The maternal blood ethanol concentration produced by the ethanol regimen was 71 +/- 12 mM. Adult offspring were killed on postnatal day 61, and cerebral cortical tissue was analyzed for ionotropic glutamate receptor subunit expression by Western immunoblotting. RESULTS: There was a statistically significant decrease in NR2B subunit protein expression and an increase in GluR2/3 subunit protein expression in the ethanol group. Expression of NR1, NR2A, NR2C, GluR1, GluR6/7, and KA2 subunit proteins was not affected. CONCLUSIONS: These results demonstrate that chronic prenatal ethanol exposure produces long-lasting effects on the subunit composition of NMDA and AMPA receptors in the cerebral cortex of the adult guinea pig.

Animals↗
PubMed2003

Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P.

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.

Base Sequence↗
PubMed2003

Receptor- and nucleotide exchange-independent mechanisms for promoting G protein subunit dissociation.

Mechanisms for heterotrimeric G protein activation that do not rely on G protein coupled receptor activation are becoming increasingly apparent. We recently identified beta gamma subunit-binding peptides that we proposed bound to a "hot spot" on beta gamma subunits, stimulating G protein dissociation without stimulating nucleotide exchange and activating G protein signaling in intact cells. AGS3, a member of the activators of G protein signaling family of proteins, also activates G protein signaling in a nucleotide exchange-independent manner, and AGS3 homologues are involved in asymmetric cell division during development. Here we demonstrate that a consensus G protein regulatory (GPR) peptide from AGS3 and related proteins is sufficient to induce G protein subunit dissociation and that both the GPR and hot spot-binding peptides promote dissociation to extents comparable with a known G protein activator, AMF. Peptides derived from adenylyl cyclase 2 and GRK2 prevented formation of the heterotrimeric complex but did not alter the rate of alpha subunit dissociation from beta gamma subunits. These data indicate that these nucleotide exchange-independent G protein activator peptides do not simply compete for alpha interactions with beta gamma subunits, but actively promote subunit dissociation. Thus, we propose two novel mechanisms for nucleotide exchange independent activation of G protein signaling, one that involves conformational changes in the alpha subunit and one that involves conformational changes in the beta gamma subunits.

Amino Acid Sequence↗
PubMed2001

Chronic prenatal ethanol exposure increases GABA(A) receptor subunit protein expression in the adult guinea pig cerebral cortex.

Excessive consumption of ethanol during pregnancy can produce teratogenic effects in offspring and is the leading cause of mental deficiency in the Western world. The objective of this study was to examine the effects of chronic prenatal ethanol exposure on the number of GABA(A) receptors and relative protein levels for GABA(A) receptor alpha1 and beta2/3 subunits in the adult guinea pig cerebral cortex. Timed pregnant Dunkin-Hartley strain guinea pigs were given one of the following oral treatments daily throughout gestation: 4 gm of ethanol per kilogram of maternal body weight, isocaloric-sucrose with pair feeding, or isovolumetric water with ad libitum access to food. The ethanol treatment resulted in a peak maternal blood ethanol concentration of 328 +/- 55 mg/dl (71.3 +/- 12.0 mm) on gestational day 57 (term, approximately 68 d). Chronic prenatal exposure to ethanol resulted in increased spontaneous locomotor activity throughout development and decreased cerebral cortical weight in adult offspring. The number of cerebral cortical [(3)H]muscimol binding sites was increased in adult offspring from the ethanol treatment group, and there was a corresponding increase in the amount of GABA(A) receptor alpha1 and beta2/3 subunit proteins in these same animals. For individual offspring, there were correlations between locomotor activity and cerebral cortical weight, as well as between cerebral cortical weight and GABA(A) receptor neurochemistry. There was no effect of chronic prenatal ethanol exposure on [(3)H]MK-801 binding in this tissue. These data demonstrate that chronic prenatal ethanol exposure has long-term consequences on the regulation of GABA(A) receptor expression in the cerebral cortex.

Administration, Oral↗
PubMed2000

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.

Adrenergic alpha-Agonists↗
PubMed2006

Differential association of protein subunits with the human RNase MRP and RNase P complexes.

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.

Cell Line, Tumor↗
PubMed2005

G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits.

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells produced a significant reduction of the extracellular signal-regulated kinase (ERK) response to CCL2. This effect is independent of its role in receptor phosphorylation because the kinase-deficient mutant GRK2K220R was able to reduce this response, and ERK activation by CCR2BIX, a phosphorylation-defective receptor mutant, was also inhibited by GRK2. Constructs containing the Galpha(q)-binding RGS-like RH domain of GRK2 or its Gbetagamma-binding domain could not reproduce the inhibition, thus revealing that GRK2 acts downstream of G proteins. Interestingly, chemokine-driven mitogen-activated protein kinase kinase (MEK) stimulation is not affected in cells overexpressing GRK2 or GRK2K220R or in splenocytes from heterozygous GRK2 mice, where reduced kinase levels correlate with enhanced ERK activation by chemokines. We find GRK2 and MEK in the same multimolecular complex, thus suggesting a mechanism for GRK2 regulation of ERK activity that involves a direct or coordinate interaction with MEK. These results suggest an important role for GRK2 in the control of chemokine induction of ERK activation at the level of the MEK-ERK interface.

Animals↗