Cloning, expression analysis and chromosome mapping of human casein kinase 1 gamma1 (CSNK1G1): identification of two types of cDNA encoding the kinase protein associated with heterologous carboxy-terminal sequences

Casein kinase 1 gamma1(CK1 gamma1) is known to be involved in the growth and morphogenesis of eukaryotic cells. We have isolated two types of cDNA for human casein kinase 1 gamma1 (hCK1 gamma1). One of them (hCK1 gamma1S) was found to encode a polypeptide consisting of 393 amino acids, which is highly homologous with already reported rat CK1 gamma1 (rCK1 gamma1). The other type of cDNA (hCK1 gamma1L) encodes a polypeptide consisting of 422 amino acids, which is quite identical in the kinase domain, but different in the C-terminal sequence from hCK1 gamma1S. Namely, hCK1 gamma1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the casein kinase gamma2 and gamma3 from rat and human, suggesting that it is a signature sequence of the gamma-isoforms. In this sense, newly isolated hCK1 gamma1L might be the original form of CK1 gamma1 subspecies rather than rCK1 gamma1 and hCK1 gamma1S. RT-PCR analysis revealed that hCK1 gamma1S mRNA is predominantly present in the testis, whereas the abundance of hCK1 gamma1L mRNA was nearly the same in the twelve tissues examined. These results suggest that novel hCK1 gamma1L may have a unique functional role different from that of hCK1 gamma1S and rCK1 gamma1. The human hCK1 gamma1 gene (CSNK1G1) was mapped to chromosome 15q22.1-->q22.31 by fluorescence in situ hybridization.     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.     

Electrophysiological evidence for the coexistence of alpha1 and alpha6 subunits in a single functional GABA(A) receptor

The subunit combinations alpha1beta2gamma2, alpha6beta2gamma2, and alpha1alpha6beta2gamma2 of the GABA(A) receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration-response curve of the channel agonist GABA for alpha1alpha6beta2gamma2 showed a single apparent component characterized by an EC(50) of 107 +/- 26 microM (n = 4). It was different from the one for alpha1beta2gamma2, which had an EC(50) of 41 +/- 9 microM (n = 4), that for alpha6beta2gamma2, with an EC(50) of 6.7 +/- 1.9 microM (n = 5), and those for alpha1beta2 and alpha1alpha6beta2. There was no appreciable functional expression of alpha6beta2. Allosteric responses of alpha1alpha6beta2gamma2 to diazepam were intermediate to those of alpha1beta2gamma2 and alpha6beta2gamma2, and allosteric responses to flumazenil were comparable to the ones for alpha1beta2gamma2. The inhibition by furosemide of the currents elicited by GABA in alpha1alpha6beta2gamma2 [IC(50) = 298 +/- 116 microM (n = 7), assuming only one component] was not identical with inhibition of alpha6beta2gamma2 (IC(50) = 38 +/- 2 microM, n = 4), alpha1beta2gamma2 (IC(50) = 5,610 +/- 910 microM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABA(A) receptors containing two different alpha subunits, alpha1 and alpha6, with properties different from those of alpha1beta2gamma2 and alpha6beta2gamma2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations alphabeta do not form in the presence of cRNA coding for the gamma subunit.     

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Inclining the purine base binding plane in protein kinase CK2 by exchanging the flanking side-chains generates a preference for ATP as a cosubstrate

Protein kinase CK2 (casein kinase 2) is a highly conserved and ubiquitously found eukaryotic serine/threonine kinase that plays a role in various cellular key processes like proliferation, apoptosis and circadian rhythm. One of its prominent biochemical properties is its ability to use GTP as well as ATP as a cosubstrate (dual-cosubstrate specificity). This feature is exceptional among eukaryotic protein kinases, and its biological significance is unknown. We describe here a mutant of the catalytic subunit of protein kinase CK2 (CK2alpha) from Homo sapiens (hsCK2alpha) with a clear and CK2-atypical preference for ATP compared to GTP. This mutant was designed on the basis of several structures of CK2alpha from Zea mays (zmCK2alpha) in complex with various ATP-competitive ligands. A structural overlay revealed the existence of a "purine base binding plane" harbouring the planar moiety of the respective ligand like the purine base of ATP and GTP. This purine base binding plane is sandwiched between the side-chains of Ile66 (Val66 in hsCK2alpha) and Met163, and it adopts a significantly different orientation than in prominent homologues like cAMP-dependent protein kinase (CAPK). By exchanging these two flanking amino acids (Val66Ala, Met163Leu) in hsCK2alpha(1-335), a C-terminally truncated variant of hsCK2alpha, the cosubstrate specificity shifted in the expected direction so that the mutant strongly favours ATP. A structure determination of the mutant in complex with an ATP-analogue confirmed the predicted change of the purine base binding plane orientation. An unexpected but in retrospect plausible consequence of the mutagenesis was, that the helix alpha D region, which is in the direct neighbourhood of the ATP-binding site, has adopted a conformation that is more similar to CAPK and less favourable for binding of GTP. These findings demonstrate that CK2alpha possesses sophisticated structural adaptations in favour of dual-cosubstrate specificity, suggesting that this property could be of biological significance.     

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.     

Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2alpha

Amino acid residues associated with functional specificity of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK-like kinases (CLKs), which are collectively termed the CMGC group, were identified by categorizing and quantifying the selective constraints acting upon these proteins during evolution. Many constraints specific to CMGC kinases correspond to residues between the N-terminal end of the activation segment and a CMGC-conserved insert segment associated with coprotein binding. The strongest such constraint is imposed on a "CMGC-arginine" near the substrate phosphorylation site with a side chain that plays a role both in substrate recognition and in kinase activation. Two nearby buried waters, which are also present in non-CMGC kinases, typically position the main chain of this arginine relative to the catalytic loop. These and other CMGC-specific features suggest a structural linkage between coprotein binding, substrate recognition, and kinase activation. Constraints specific to individual subfamilies point to mechanisms for CMGC kinase specialization. Within casein kinase 2alpha (CK2alpha), for example, the binding of one of the buried waters appears prohibited by the side chain of a leucine that is highly conserved within CK2alpha and that, along with substitution of lysine for the CMGC-arginine, may contribute to the broad substrate specificity of CK2alpha by relaxing characteristically conserved, precise interactions near the active site. This leucine is replaced by a conserved isoleucine or valine in other CMGC kinases, thereby illustrating the potential functional significance of subtle amino acid substitutions. Analysis of other CMGC kinases similarly suggests candidate family-specific residues for experimental follow-up.     

Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2

The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system. CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.     

Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of the CK2alpha'-based holoenzyme, an isozyme of CK2alpha: a comparative analysis

Protein kinase CK2 (former name: "casein kinase 2") is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2alpha) and two regulatory subunits (CK2beta). In higher animals two paralog catalytic chains-abbreviated CK2alpha and CK2alpha'--exist which can combine with CK2beta to three isoforms of the holoenzyme: CK2alpha(2)beta(2), CK2alpha(2)(')beta(2), and CK2alphaalpha(')beta(2). While CK2alpha and the "normal" holoenzyme CK2alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2alpha' and the "alternative" holoenzyme CK2alpha(2)(')beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2alpha' rather than CK2alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2alpha(2)(')beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2alpha'-MBP in the presence of human CK2beta so that CK2alpha' subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2alpha'-based and the CK2alpha-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.     

2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein kinase 1 gamma (CK1γ) inhibitors

Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.     

Specific activation of leukocyte beta2 integrins lymphocyte function-associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains.     

Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the gamma 2 subunit.

The mammalian GABAA receptor is a multisubunit protein containing a variety of binding sites for psychotropic agents. One of the most widely used of these drugs, ethanol, enhances the function of GABAA receptors in certain circumstances but not others. Previous studies have demonstrated that alternative splicing of the gamma 2L GABA subunit results in an ethanol sensitive and an ethanol-insensitive form, when combined with alpha and beta subunits. We have used in vitro mutagenesis and expression in Xenopus oocytes to show that the consensus site for phosphorylation by protein kinase C contained in the gamma 2L insert is critical for modulation by ethanol but not benzodiazepines, and manipulation of the phosphorylating enzymes in oocytes containing alpha 1 beta 1 gamma 2L can prevent ethanol enhancement. It is likely that phosphorylation or dephosphorylation of a specific site on the GABAA receptor protein can act as a control mechanism for neuronal responses to alcohol exposure.     

The metabotropic glutamate receptor mGluR7b binds to the catalytic gamma-subunit of protein phosphatase 1

Correct targeting of enzymes represents an important biological mechanism to control post-translational modifications of neurotransmitter receptors. The metabotropic glutamate receptor type 7 (mGluR7) exists in two splice variants (mGluR7a and mGluR7b), defined by different C-termini that are phosphorylated by protein kinase C (PKC). Recently, the search for mGluR7a binding partners yielded several proteins that interacted with its C-terminus. Here, a yeast two-hybrid screen using the mGluR7b C-terminus identified both variants of the catalytic gamma-subunit of protein phosphatase 1 (PP1gamma1 and PP1gamma2) as binding partners. The minimal interacting region of PP1gamma1/2 contained the core domain and was homologous to a region of PP1alpha that is needed for functional expression. Although this core domain is highly conserved within the protein phosphatase family, PP1alpha1 and PP1beta did not interact with mGluR7b. Binding between PP1gamma1 and mGluR7b might be regulated by alternative splicing, as the variant-specific distal part of the mGluR7b C-terminus mediated the interaction. Within this domain, amino acids involved in the binding to PP1gamma1 were mapped and biochemical assays using recombinant and native proteins verified the proposed interaction. Finally, the expression pattern of PP1gamma1, PP1gamma2 and mGluR7b was analysed in various CNS regions. In summary, these results suggest a regulation of mGluR7b by PP1gamma.     

Point mutations affecting antagonist affinity and agonist dependent gating of GABAA receptor channels.

Two variant amino acid sequences, which differ in a single amino acid residue, have been reported for the alpha 1-subunit of the rat brain GABAA receptor. We separately co-expressed these two variants in Xenopus oocytes, in combination with beta 2 and gamma 2. This experiment showed that substitution of alpha 1-Phe64 by Leu strongly decreases the apparent affinity for GABA dependent channel gating from 6 microM to 1260 microM. Starting from this observation, we used in vitro mutagenesis to obtain information relevant for the localization of the agonist/antagonist binding site in the GABAA receptor. Homologous mutation in alpha 5 had similar consequences for alpha 5 beta 2 gamma 2. Homologous mutation in beta 2 and gamma 2 resulted in intermediate and small shifts in EC50, respectively. The apparent affinities of the competitive antagonists bicuculline methiodide and SR95531, the latter sharing close structural similarity with the agonist GABA, were decreased 60- to 200-fold by these mutations in alpha-subunits. Interestingly, these affinities remained nearly unaffected upon introduction of the homologous mutations in beta 2 and gamma 2, or upon mutation of the neighbouring amino acid in alpha 1, Phe65 to Leu. These results suggest close functional and structural association of alpha-subunits with the agonist/antagonist binding site, and involvement of N-terminal portions of the extracellular domains of all subunits in the gating of the channel.