Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Motif B loop”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed1999

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily.

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.

Adenosine Triphosphatases↗
PubMed2002

Human immune associated nucleotide 1: a member of a new guanosine triphosphatase family expressed in resting T and B cells.

TAL-1 is a basic helix-loop-helix oncoprotein that is expressed in up to 30% of T-cell acute lymphoblastic leukemias but not in the T lineage. We have cloned a complementary DNA, called Human Immune Associated Nucleotide 1 (hIAN1), whose messenger RNA (mRNA) level expression is inversely correlated to the TAL-1 mRNA level in human leukemic T-cell lines. The hIAN1 encodes a 38-kd protein that belongs to a novel family of proteins conserved from plants to humans and characterized by motifs related to, but highly divergent from, the consensus motifs found in guanosine triphosphate (GTP)-binding proteins. Despite these divergent amino acids at positions involved in GTP/guanosine diphosphate (GDP) binding and guanosine triphosphatase (GTPase) activities, we found that hIAN1 specifically binds GDP (K(d) = 0.47 microM) and GTP (K(d) = 6 microM) and exhibits intrinsic GTPase activity. Among mature hematopoietic cells, hIAN1 is specifically expressed in resting T and B lymphocytes, and its expression level tremendously decreased at the protein but not the mRNA level during B- or T-lymphocyte activation, suggesting a specific role for this new type of GTPase during the immune response.

Amino Acid Sequence↗
PubMed1996

Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ.

The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% alpha helix, 21% beta sheet, and 45% other secondary structure. Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD [Rost, B., & Sander, C. (1993a) J. Mol. Biol. 232, 584-599]. The predicted secondary structures average of 33% alpha helix, 24% beta sheet, and 43% loop for the alpha beta dimer. The predictions have been compared with sites of limited proteolysis by 12 proteases at the surfaces of the heterodimer and taxol-induced microtubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry 35, 14184-14202]. From 24 experimentally determined nicking sites, 18 are at predicted loops or at the extremes of secondary structure elements. Proteolysis zone A (including acetylable Lys40 and probably Lys60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules. Proteolysis zone C, between residues 278 and 295, becomes partially occluded in microtubules. The alpha-tubulin nicking site Arg339-Ser340 is at a loop following a predicted alpha helix in proteolysis zone D. This site is protected in taxol microtubules; however, a new tryptic site appears which is probably located at the N-terminal end of the same helix. Zone D also contains beta-tubulin Cys354, which is accessible in microtubules. Proteolysis zone E includes the C-terminal hypervariable loops (10-20 residues) of each tubulin chain. These follow the two larger predicted helical zones (residues 372-395 and 405-432 in beta-tubulin), which also are the longer conserved part of the alpha- and beta-tubulin sequences. Through combination of this with other biochemical information, a set of surface and distance constraints is proposed for the folding of beta-tubulin. The FtsZ sequences are only 10-18% identical to the tubulin sequences. However, the predicted secondary structures show two clearly similar (85-87 and 51-78%) regions, at tubulin positions 95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300, respectively. The first region is flanked by tubulin proteolysis zones A and B. It consists of a predicted loop1-helix-loop2-sheet-loop3-helix-loop4-sheet fold, which contains the motif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif (SAG)GGTG(SAT)G (loop3). A simple working model envisages loop1 and loop3 together at the nucleotide binding site, while loops 2 and 4 are at the surface of the protein, in agreement with proteolytic and antigenic accessibility results in tubulin. The model is compatible with studies of tubulin and FtsZ mutants. It is proposed that this region constitutes a common structural and evolutionary nucleus of tubulins and FtsZ which is different from typical GTPases.

Amino Acid Sequence↗
PubMed1993

Genetic heterogeneity of the V3 region of the HIV-1 envelope glycoprotein in Brazil. Brazilian Collaborative AIDS Research Group.

OBJECTIVE: To examine the genetic heterogeneity of the V3 region of HIV-1 gp120 from 22 Brazilian HIV-1 specimens. DESIGN: Genetic heterogeneity was examined by DNA sequencing of the C2 V3 region of the HIV-1 envelope (env) gene from polymerase chain reaction (PCR)-amplified HIV-1 DNA. Deduced amino-acid sequences were compared to determine the extent of amino-acid conservation among the Brazilian specimens. Genetic similarity among and between the Brazilian specimens and other previously published HIV-1 isolates was analyzed by principal co-ordinate and DNA parsimony methods. METHODS: A 282 base pair (bp) region of a 1.5 kilo (k) bp PCR-amplified HIV-1 env fragment was sequenced by a Taq dye-labeled primer cycle sequencing reaction. Nucleotide sequences were used to analyze inter-specimen relationships based on overall nucleotide sequence similarity and DNA parsimony principles. RESULTS: Amino-acid comparison showed that 15 of the 35 (43%) residues of the V3 loop were conserved among the Brazilian specimens. Nine of the 22 (40%) Brazilian specimens contained the North American-European GPGR tetrapeptide motif, while eight (36%) contained the GWGR motif, previously reported in Japanese isolates. Principal co-ordinate analysis demonstrated that 19 of the 20 examined Brazilian HIV-1 specimens were more similar to North American and Haitian isolates than to African isolates. Similar results were also obtained by DNA parsimony analysis. CONCLUSION: The majority of the Brazilian specimens examined are more genetically related to North American and Haitian HIV-1 isolates than to African isolates. This finding and the presence of a GWGR V3 loop motif in some Brazilian isolates may be important for vaccine development.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Act1, an NF-kappa B-activating protein.

Use of an NF-kappaB-dependent selectable marker facilitated the isolation of a cell line containing a cDNA encoding Act1, an NF-kappaB activator. Act1 associates with and activates IkappaB kinase (IKK), leading to the liberation of NF-kappaB from its complex with IkappaB. Many signaling pathways that liberate NF-kappaB also activate activating transcription factor (ATF) and activator protein 1 (AP-1) through Jun kinase (JNK). Act1 also activates JNK, suggesting that it might be part of a multifunctional complex involved in the activation of both NF-kappaB and JNK. Act1 fails to activate NF-kappaB in an IL-1-unresponsive mutant cell line in which all known signaling components are present, suggesting that it interacts with an unknown component in IL-1 signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8.

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.

Amino Acid Motifs↗
PubMed1996

The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription.

The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 genes is critical for B-cell leukemogenesis.

Base Sequence↗
PubMed1999

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation.

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.

Amino Acid Sequence↗
PubMed1991

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.

Adult↗
PubMed2001

DNA binding and transcriptional activation by a PDX1.PBX1b.MEIS2b trimer and cooperation with a pancreas-specific basic helix-loop-helix complex.

In pancreatic acinar cells, the HOX-like factor PDX1 acts as part of a trimeric complex with two TALE class homeodomain factors, PBX1b and MEIS2b. The complex binds to overlapping half-sites for PDX1 and PBX. The trimeric complex activates transcription in cells to a level about an order of magnitude greater than PDX1 alone. The N-terminal PDX1 activation domain is required for detectable transcriptional activity of the complex, even though PDX1 truncations bearing only the PDX1 C-terminal homeodomain and pentapeptide motifs can still participate in forming the trimeric complex. The conserved N-terminal PBC-B domain of PBX, as well as its homeodomain, is required for both complex formation and transcriptional activity. Only the N-terminal region of MEIS2, including the conserved MEIS domains, is required for formation of a trimer on DNA and transcriptional activity: the MEIS homeodomain is dispensable. The activity of the pancreas-specific ELA1 enhancer requires the cooperation of the trimer-binding element and a nearby element that binds the pancreatic transcription factor PTF1. We show that the PDX1. PBX1b.MEIS2b complex cooperates with the PTF1 basic helix-loop-helix complex to activate an ELA1 minienhancer in HeLa cells and that this cooperation requires all three homeoprotein subunits, including the PDX1 activation domain.

Cell Line↗
PubMed1995

The E12 inhibitory domain prevents homodimer formation and facilitates selective heterodimerization with the MyoD family of gene regulatory factors.

Although the ubiquitous helix-loop-helix (HLH) protein E12 does not homodimerize efficiently, the myogenic factor MyoD forms an avid DNA-binding heterodimer with E12 through the conserved HLH dimerization domain. However, the mechanism which ensures this selective dimerization is not understood at present. In our functional studies of various amino acid changes in the E12 HLH domain, we found that a single substitution in E12 helix 1 can abolish the effect of the E12 inhibitory domain and results in the efficient DNA binding of the E12 homodimer. Competition experiments revealed that the inhibitory domain, in fact, blocks the dimerization of E12 rather than DNA binding. MyoD contains two glutamic residues in helix 2 that are required for efficient dimerization with E12. More importantly, these residues were not essential for dimerization with E12 mutants in which the dimerization inhibitory domain had been relaxed, or for dimerization with E47 which does not contain the inhibitory domain owing to the use of an alternative exon. The positions of these glutamic residues are conserved among the four myogenic factors. Thus, members of the MyoD family of gene regulatory proteins can overcome the E12 dimerization inhibitory domain through a mechanism involving, in part, the negatively charged amino acid residues in helix 2. This result describes a novel mechanism facilitating the selective formation of the MyoD(MRF)-E12 heterodimer that enhances dimerization specificity and may apply to other members of the E-protein family.

3T3 Cells↗
PubMed2003

Atomic resolution structure of obelin: soaking with calcium enhances electron density of the second oxygen atom substituted at the C2-position of coelenterazine.

The spatial structure of the Ca(2+)-regulated photoprotein obelin has been solved to resolution of 1.1A. Two oxygen atoms are revealed substituted at the C2-position of the coelenterazine in contrast to the obelin structure at 1.73A resolution where one oxygen atom only was disclosed. The electron density of the second oxygen atom was very weak but after exposing the crystals to a trace of Ca(2+), the electron densities of both oxygen atoms became equally intense. In addition, one Ca(2+) was found bound in the loop of the first EF-hand motif. Four of the ligands were provided by protein residues Asp30, Asn32, Asn34, and the main chain oxygen of Lys36. The other two were from water molecules. From a comparison of B-factors for the residues constituting the active site, it is suggested that the variable electron densities observed in various photoprotein structures could be attributed to different mobilities of the peroxy oxygen atoms.

Binding Sites↗
PubMed1997

MyoD-E12 heterodimers and MyoD-MyoD homodimers are equally stable.

Muscle development is controlled by the MyoD family of basic helix-loop-helix (bHLH) DNA-binding proteins. These proteins dimerize with ubiquitous products of the E2A gene (E12 and E47) and bind in a sequence-specific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been utilized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociation (deltaG) and the equilibrium dissociation constants (K(D)) for the protein-protein interactions are reported. The deltaG for the MyoD homodimers in 100 mM KCl was 8.7 kcal/mol (K(D) = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigorous confidence limit testing. The titrations of E12-dansyl with MyoD yielded a free energy of 8.3 kcal/mol with tighter confidence limits, +0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers and MyoD-E12 heterodimers are relatively weak complexes of approximately the same stability. E12 does not form stable homo-oligomeric complexes; remaining monomeric at concentrations as high as 20 microM. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterodimers versus MyoD homodimers with DNA binding sites is not due to preferential heterodimerization.

Animals↗
PubMed1993

Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues.

The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with E12. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with E12. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and E12 and determine the dimerization specificity of the bHLH proteins.

Amino Acid Sequence↗
PubMed1994

Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells.

The terminal differentiation of mammalian muscle cells requires the tumor suppressor retinoblastoma protein (Rb). Unlike their wild-type counterparts, multinucleated myotubes from mouse cells deficient in Rb (Rb-/-) were induced by serum to re-enter the cell cycle. Development of the myogenic phenotype in Rb-/- cells correlated with increased expression of p107, which interacted with myogenic transcription factors. Serum-induced cell cycle reentry, on the other hand, correlated with decreased p107 expression. Thus, although p107 could substitute for Rb as a cofactor for differentiation, it could not maintain the terminally differentiated state in Rb-/- myotubes.

Animals↗
PubMed2000

Overlapping expression of early B-cell factor and basic helix-loop-helix proteins as a mechanism to dictate B-lineage-specific activity of the lambda5 promoter.

The basic helix-loop-helix (bHLH) transcription factors are a large group of proteins suggested to control key events in the development of B lymphocytes as well as of other cellular lineages. To examine how bHLH proteins activate a B-lineage-specific promoter, I investigated the ability of E47, E12, Heb, E2-2, and MyoD to activate the lambda5 surrogate light chain promoter. Comparison of the functional capacity of the E2A-encoded E47 and E12 proteins indicated that even though both were able to activate the lambda5 promoter and act in synergy with early B-cell factor (EBF), E47 displayed a higher functional activity than E12. An ability to act in synergy with EBF was also observed for Heb, E2-2, and MyoD, suggesting that these factors were functionally redundant in this regard. Mapping of functional domains in EBF and E47 revealed that the dimerization and DNA binding domains mediated the synergistic activity. Electrophoretic mobility shift assay analysis using the 5' part of the lambda5 promoter revealed formation of template-dependent heteromeric complexes between EBF and E47, suggesting that the synergistic mechanism involves cooperative binding to DNA. These findings propose a unique molecular function for E47 and provide overlapping expression with EBF as a molecular mechanism to direct B-cell-specific target gene activation by bHLH proteins.

B-Lymphocytes↗
PubMed1994

Purification of a recombinant Schistosoma japonicum antigen homologous to the 22-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis.

We describe the cDNA cloning, overproduction and purification of a 22.6-kDa antigen from the human blood fluke Schistosoma japonicum. A 777-bp cDNA (C32) was isolated from a S. japonicum lambda ZAPII cDNA expression library immuno-screened with hyperimmune rabbit serum (HRS) raised against soluble adult S. japonicum proteins. The open reading frame of C32 encodes a protein of 191 amino acids (aa) which exhibits 71% identity to a 22.6-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate for schistosomiasis. We have identified a sequence motif known as an EF-hand calcium-binding domain in both the S. japonicum and S. mansoni aa sequences, suggesting that the 22.6-kDa antigens are able to bind Ca2+. Further, we have, for the first time, obtained the 22.6-kDa antigen in purified, non-denatured, recombinant form, and in sufficient quantity to assess the protective value of the molecule in vaccination/challenge experiments. This was achieved by synthesizing the schistosome antigen with a short polyhistidine tag fused to the N-terminus which was then used for subsequent affinity purification. The recombinant protein was purified under non-denaturing conditions using nickel-chelate affinity chromatography.

Amino Acid Sequence↗
PubMed1995

Mutation of two conserved arginine residues in the glucose transporter GLUT4 supresses transport activity, but not glucose-inhibitable binding of inhibitory ligands.

Two arginine residues (RR333/334) in the conserved GRR motif located in the endofacial loop between helix 8 and 9 of the glucose transporter GLUT4 were substituted for leucine and alanine, respectively. Reconstituted glucose transport activity of the construct (GLUT4-RR333/4LA) expressed in COS-7 or LM(TK-) cells was less than 10% of that of the wild-type GLUT4. In contrast, binding of the inhibitory ligand cytochalasin B and glucose-inhibitable photolabeling with IAPS-forskolin were not significantly affected. Exchange of a histidine residue (H337Q) previously believed to be involved in the binding of inhibitory ligands failed to affect any of the investigated parameters. These data suggest that positive charges in the GRR motif at the cytoplasmic surface of the transporter participate in the conformational changes of the carrier protein during the process of facilitated diffusion.

Animals↗