Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based on amino acid sequence and computer modeling, two conflicting three-dimensional models of the dopamine D2 receptor have been proposed. One model (Dahl et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8111) suggests that dopamine interacts with aspartate 80 of transmembrane (TM) 2 and asparagine 390 of TM6 with the transmembranes arranged in a clockwise manner, while a second model (Hibert et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopamine interacts with aspartate 114 of TM3 and the serines of TM5 (194 and 197) with the transmembranes arranged in a counterclockwise manner when viewed from the extracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and 197 of the human dopamine D2 receptor by site-directed mutagenesis. In addition, two methionines (116 and 117) were mutated to evaluate whether residues near aspartate (114) of the dopamine D2 receptor are critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartate (114) to either asparagine or glycine led to a total loss of both agonist and antagonist binding. Individual or dual methionine mutations in positions 116 and 117, to make the dopamine D2 binding pocket more closely resemble the beta 2-adrenoceptor, did not result in a change in selectivity toward noradrenergic agonists or antagonists. The serine mutations revealed interesting differences between the dopamine D2 receptor and the adrenoceptors. In particular, serine 197 appeared more important than serine 194 for agonist binding. In addition, the binding of one agonist (N-0437) was unaffected by individual serine mutations, while the binding of some antagonists, such as raclopride and spiperone, was significantly altered. These findings are discussed in relation to ligand structure and their interactions with the putative binding pocket.
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PMID:Site-directed mutagenesis of the human dopamine D2 receptor. 135 63

Non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant (3)JHNHalpha and integration of the nuclear Overhauser HN-Halpha cross peak, positive theta-angles could be determined reliably to 60 degrees +/- 30 degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive theta-angles can also occur in non-glycine residues and in the four proteins, positive theta-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured (3)JHNHalpha coupling constants and the intensity of the intraresidue HN-Halpha NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wuthrich, K. (1984) J. Mol. Biol., 180, 741-751; Ludvigsen, S. Andersen, K.V. and Poulsen, F.M. (1991) J Mol. Biol., 217, 731-736).
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PMID:Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy. 139 67

Three u.v.-induced mutants of the purine-cytosine permease gene of Saccharomyces cerevisiae, with altered apparent Michaelis constant of transport (Kmapp), were cloned and sequenced. One of the mutants had extensive nucleotide replacement, whereas the other two had a single mutation. To evaluate the contribution of the different amino acid replacements to the phenotype of the complex mutant, simpler mutants were created by site-directed mutagenesis. All the amino acid replacements found in the segment from amino acids 371 to 377 inclusive, contribute to the determination of the phenotype. According to the model postulated this segment lies on the cell surface. In particular, amino acids at position 374 and 377 modulate the affinity of the permease towards its substrates. In the wild-type, when asparagine is present at both of these positions, the lowest Kmapp values are found.
Mol Microbiol 1992 Oct
PMID:Determination of a specific region of the purine-cytosine permease involved in the recognition of its substrates. 147 90

The T7 polymerase transcription system was used for in vitro synthesis of unmodified versions of the E. coli tRNA mutants that insert asparagine, cysteine, glycine, histidine, and serine. These tRNAs were used to qualitatively explore the role of some anticodon bases and the discriminator nucleotide in the recognition of tRNA by aminoacyl-tRNA synthetases. Coupled with data from earlier studies, these new results essentially complete a survey of all E. coli tRNAs with respect to the involvement of anticodon bases and the discriminator nucleotide in tRNA recognition. It is found that in the vast majority of tRNAs both of these elements are significant components of tRNA identity. This is not universally true, however. Anticodon sequences are unimportant in tRNA(Ser), tRNA(Leu), and tRNA(Ala) while the discriminator base is inconsequential in tRNA(Ser) and tRNA(Thr). The significance of these results for origin-of-life studies is discussed.
J Mol Evol 1992 Nov
PMID:The role of anticodon bases and the discriminator nucleotide in the recognition of some E. coli tRNAs by their aminoacyl-tRNA synthetases. 148 27

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.
Mol Immunol 1992 Sep
PMID:The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes. 149 1

Testicular androgen-binding protein (ABP) and liver sex hormone-binding globulin are encoded by the same gene. These proteins have the same primary amino acid sequences, but they differ in attached oligosaccharides; the differences are presumably due to cell-specific glycosylation mechanisms. To investigate the role of oligosaccharides in ABP/sex hormone-binding globulin subunit structure, secretion, and steroid binding, mutant rat ABP proteins were constructed that eliminated one or both of the two potential sites of asparagine (Asn)-linked glycosylation. Immunoblot analysis of wild type recombinant ABP yielded the typical heterogeneous banding pattern. Secreted ABP was composed of two protomers of M(r) 46,000 and M(r) 43,000, while cellular ABP yielded three mol wt species (M(r) 43,000, 41,000, and 39,000). Substitution of the Asn residue in either consensus sequence for Asn-linked glycosylation with an Ile residue resulted in increased mobility of the immunoreactive ABP species. These changes are consistent with the loss of an Asn-linked oligosaccharide. Substitution of both Asn residues yielded a single immunoreactive species in the medium and cell extracts that migrated as a M(r) 39,000 protein. These results demonstrate that the mol wt heterogeneity of ABP is due to differential Asn-linked glycosylation of both potential sites. All three mutant forms of ABP were secreted by the COS cells. However, the amount of immunoreactive ABP and [3H]5 alpha-dihydrotestosterone binding in the medium was lower than wild type (100%) in one of the single mutants (65%) and in the double mutant (29%). Unlike the glycosylation mutants, alteration of other residues, not involved in glycosylation, yielded cellular ABP and no detectable medium ABP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Jul
PMID:The role of asparagine-linked oligosaccharides in the subunit structure, steroid binding, and secretion of androgen-binding protein. 150 25

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.
J Mol Biol 1992 Sep 05
PMID:Site-directed mutagenesis of the GTP-binding domain of beta-tubulin. 152 95

Using homologous probes for the cloning of related genes within the family of guanine nucleotide-binding protein-coupled receptors, we have cloned the gene for the rhesus macaque D1 dopamine receptor. By using the rat D1 receptor coding sequence as a probe under high stringency conditions, the rhesus D1 receptor gene was isolated from a lambda EMBL3 rhesus genomic DNA library. The rhesus D1 dopamine receptor gene is intronless and encodes a 446-amino acid protein that contains two consensus sites for asparagine-linked glycosylation (Asn-5 and Asn-176) and two consensus sites for cAMP-dependent protein kinase phosphorylation (Thr-136 and Thr-268). The primary amino acid sequence of the rhesus D1 dopamine receptor shows an extremely high degree of similarity (99.6%) to the human D1 receptor. Genomic DNA analyses conducted with high and reduced stringency hybridizations indicate that the rhesus macaque D1 receptor is a member of a large multigene family. Like the human D1 receptor mRNA, the rhesus D1 receptor mRNA is approximately 4 kilobases in size and is localized predominantly in the caudate, with lesser amounts in the hippocampus and cortex. The rhesus D1 receptor coding region was inserted into the cytomegalovirus promoter-driven expression vector pcDNA-1, and the recombinant (pcDNA-D1) was cotransfected with the selectable marker pRSVneo, conferring G418 resistance, into D1 receptor-deficient C6 glioma cells. Analyses of the selected transfectant demonstrate the expression of a high affinity, functional D1 dopamine receptor. The D1 receptor radioligand [3H]SCH 23390 bound transfectant membranes with an affinity (Kd), of 0.3 nM; the D2-selective ligand spiperone, the dopamine receptor ligand clozapine, and the serotonin receptor antagonist ketanserin bound with considerably lower affinities (102, 80, and 95 nM, respectively). Both dopamine and the D1-selective agonist SKF 38393 inhibited the binding of [3H]SCH 23390 to transfectant cell membranes; the binding of these agonists was sensitive to GTP. Dopamine potently stimulated the accumulation of cAMP in transfected C6 cells, whereas SKF 38393 was a partial agonist in these cells. Also, the density of recombinant D1 receptors on the transfectant cells was decreased 40% upon treatment with 10 microM dopamine, indicating that occupation of recombinant D1 receptors by agonists alters surface expression of the receptors.
Mol Pharmacol 1992 Apr
PMID:Molecular cloning and expression of the rhesus macaque D1 dopamine receptor gene. 153 68

The proto-oncogene Wnt-1 encodes a cysteine-rich, secretory glycoprotein implicated in virus-induced mouse mammary cancer and intercellular signaling during vertebrate neural development. To attempt to correlate structural motifs of Wnt-1 protein with its function, 12 mutations were introduced singly and in several combinations into the coding sequence of Wnt-1 cDNA by site-directed mutagenesis. Mutant alleles in a retroviral vector were tested for their ability to transform the mouse mammary epithelial cell line C57MG in two ways: by direct infection of C57MG cells and by infection of NIH3T3 cells that serve as donors of Wnt-1 protein to adjacent C57MG cells in a secretion-dependent (paracrine) assay. In addition, the synthesis and secretion of mutant proteins were monitored in multiple cell types by immunological assays. Deletion of the signal peptide demonstrated that transformation in both direct and paracrine assays depends upon entry of Wnt-1 protein into the endoplasmic reticulum. Changes in potential proteolytic processing sites (two basic dipeptides and a probable signal peptidase cleavage site) did not adversely impair biological activity or protein processing and uncovered a second site for cleavage by signal peptidase. Replacement of each of the four asparagine-linked glycosylation sites did not affect transforming activity at normal temperatures, but one glycosylation site mutant was found to be temperature-sensitive for transformation. An allele encoding a protein that lacks all four glycosylation sites was also transformation competent. In two of four cases, substitution of serine for a cysteine residue impaired transforming activity at the usual temperature, and transformation was temperature sensitive in a third case, implying that at least some of the highly conserved cysteine residues are important for Wnt-1 function.
Mol Biol Cell 1992 May
PMID:Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line. 153 41

The X-ray structure of the periplasmic ribose receptor (binding protein) of Escherichia coli (RBP) was solved at 3 A resolution by the method of multiple isomorphous replacement. Alternating cycles of refitting and refinement have resulted in a model structure with an R-factor of 18.7% for 27,526 reflections from 7.5 to 1.7 A resolution (96% of the data). The model contains 2228 non-hydrogen atoms, including all 271 residues of the amino acid sequence, 220 solvent atoms and beta-D-ribose. The protein consists of two highly similar structural domains, each of which is composed of a core of parallel beta-sheet flanked on both sides by alpha-helices. The two domains are related to each other by an almost perfect 2-fold axis of rotation, with the C termini of the beta-strands of each sheet pointing toward the center of the molecule. Three short stretches of amino acid chain (from symmetrically related portions of the protein) link these two domains, and presumably act as a hinge to allow relative movement of the domains in functionally important conformational changes. Two water molecules are also an intrinsic part of the hinge, allowing crucial flexibility in the structure. The ligand beta-D-ribose (in the pyranose form) is bound between the domains, held by interactions with side-chains of the interior loops. The binding site is precisely tailored, with a combination of hydrogen bonding, hydrophobic and steric effects giving rise to tight binding (0.1 microM for ribose) and high specificity. Four out of seven binding-site residues are charged (2 each of aspartate and arginine) and contribute two hydrogen bonds each. The remaining hydrogen bonds are contributed by asparagine and glutamine residues. Three phenylalanine residues supply the hydrophobic component, packing against both faces of the sugar molecule. The arrangement of these hydrogen bonding and hydrophobic residues results in an enclosed binding site with the exact shape of the allowed sugar molecules; in the process of binding, the ligand loses all of its surface-accessible area. The sites of two mutations that affect the rate of folding of the ribose receptor are shown to be located near small cavities in the wild-type protein. The cavities thus allow the incorporation of the larger residues in the mutant proteins. Since these alterations would seriously affect the ability of the protein to build the first portion of the hydrophobic core in the first domain, it is proposed that this process is the rate-limiting step in folding of the ribose receptor.
J Mol Biol 1992 May 05
PMID:1.7 A X-ray structure of the periplasmic ribose receptor from Escherichia coli. 158 88


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