Search PubMed⌕ Search

Biomedical subjects

H Weinstein

Publications and source records attributed to H Weinstein.

At least 55 records · Page 3Linked to original sources
PubMed1997

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

Adrenergic beta-Agonists↗
PubMed1997

A surface on the G protein beta-subunit involved in interactions with adenylyl cyclases.

Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.

Adenylyl Cyclases↗
PubMed1997

Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.

Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.

Adrenergic beta-Agonists↗
PubMed1997

Does TATA matter? A structural exploration of the selectivity determinants in its complexes with TATA box-binding protein.

The binding of the TATA box-binding protein (TBP) to a TATA sequence in DNA is essential for eukaryotic basal transcription. TBP binds in the minor groove of DNA, causing a large distortion of the DNA helix. Given the apparent stereochemical equivalence of AT and TA basepairs in the minor groove, DNA deformability must play a significant role in binding site selection, because not all AT-rich sequences are bound effectively by TBP. To gain insight into the precise role that the properties of the TATA sequence have in determining the specificity of the DNA substrates of TBP, the solution structure and dynamics of seven DNA dodecamers have been studied by using molecular dynamics simulations. The analysis of the structural properties of basepair steps in these TATA sequences suggests a reason for the preference for alternating pyrimidine-purine (YR) sequences, but indicates that these properties cannot be the sole determinant of the sequence specificity of TBP. Rather, recognition depends on the interplay between the inherent deformability of the DNA and steric complementarity at the molecular interface.

Algorithms↗
PubMed1997

Comparative modeling and molecular dynamics studies of the delta, kappa and mu opioid receptors.

Molecular models of the trans-membrane domains of delta, kappa and mu opioid receptors, members of the G-protein coupled receptor (GPCR) superfamily, were developed using techniques of homology modeling and molecular dynamics simulations. Structural elements were predicted from sequence alignments of opioid and related receptors based on (i) the consensus, periodicities and biophysical interpretations of alignment-derived properties, and (ii) tertiary structure homology to rhodopsin. Initial model structures of the three receptors were refined computationally with energy minimization and the result of the first 210 ps of a 2 ns molecular dynamics trajectory at 300K. Average structures from the trajectory obtained for each receptor subtype after release of the initial backbone constraints show small backbone deviations, indicating stability. During the molecular dynamics phase, subtype-differentiated residues of the receptors developed divergent structures within the models, including changes in regions common to the three subtypes and presumed to belong to ligand binding regions. The divergent features developed by the model structures appear to be consistent with the observed ligand binding selectivities of the opioid receptors. The results thus implicate identifiable receptor microenvironments as primary determinants of some of the observed subtype specificities in opiate ligand binding and in functional effects of mutagenesis. Networks of interacting residues observed in the models are common to the opiate receptors and other GPCRs, indicating core interfaces that are potentially responsible for structural integrity and signal transduction. Analysis of extended molecular dynamics trajectories reveals concerted motions of distant parts of ligand-binding regions, suggesting motion-sensitive components of ligand binding. The comparative modeling results from this study help clarify experimental observations of subtype differences and suggest both structural and dynamic rationales for differences in receptor properties.

Amino Acid Sequence↗
PubMed1996

Gbeta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2. Cross-linking of the peptide to free Gbetagamma but not the heterotrimer.

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

Adenylyl Cyclases↗
PubMed1996

Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice.

The binding site of the dopamine D2 receptor, like that of other homologous G-protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.

Amino Acid Sequence↗
PubMed1996

Mapping the binding site pocket of the serotonin 5-Hydroxytryptamine2A receptor. Ser3.36(159) provides a second interaction site for the protonated amine of serotonin but not of lysergic acid diethylamide or bufotenin.

Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

Amino Acid Sequence↗
PubMed1996

Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome.

Long-term survival and improved neuropsychological function have occurred in selected children with Hurler syndrome (MPS I H) after successful engraftment with genotypically matched sibling bone marrow transplantation (BMT). However, because few children have HLA-identical siblings, the feasibility of unrelated donor (URD) BMT as a vehicle for adoptive enzyme therapy was evaluated in this retrospective study. Forty consecutive children (median, 1.7 years; range, 0.9 to 3.2 years) with MPS I H received high-dose chemotherapy with or without radiation followed by BMT between January 27, 1989 and May 13, 1994. Twenty-five of the 40 patients initially engrafted. An estimated 49% of patients are alive at 2 years, 63% alloengrafted and 37% autoengrafted. The probability of grade II to IV acute graft-versus-host disease (GVHD) was 30%, and the probability of extensive chronic GVHD was 18%. Eleven patients received a second URD BMT because of graft rejection or failure. Of the 20 survivors, 13 children have complete donor engraftment, two children have mixed chimeric grafts, and five children have autologous marrow recovery. The BM cell dose was correlated with both donor engraftment and survival. Thirteen of 27 evaluable patients were engrafted at 1 year following URD BMT. Neither T-lymphocyte depletion (TLD) of the bone marrow nor irradiation appeared to influence the likelihood of engraftment. Ten of 16 patients alive at 1 year who received a BM cell dose greater than or equal to 3.5 x 10(8) cells/kg engrafted, and 62% are estimated to be alive at 3 years. In contrast, only 3 of 11 patients receiving less than 3.5 x 10(8) cells/kg engrafted, and 24% are estimated to be alive at 3 years (P = .05). The mental developmental index (MDI) was assessed before BMT. Both baseline and post-BMT neuropsychological data were available for 11 engrafted survivors. Eight children with a baseline MDI greater than 70 have undergone URD BMT (median age, 1.5 years; range, 1.0 to 2.4 years). Of these, two children have had BMT too recently for developmental follow-up. Of the remaining six, none has shown any decline in age equivalent scores. Four children are acquiring skills at a pace equal to or slightly below their same age peers; two children have shown a plateau in learning or extreme slowing in their learning process. For children with a baseline MDI less than 70 (median age, 2.5 years; range, 0.9 to 2.9 years), post-BMT follow-up indicated that two children have shown deterioration in their developmental skills. The remaining three children are maintaining their skills and are adding to them at a highly variable rate. We conclude that MPS I H patients with a baseline MDI greater than 70 who are engrafted survivors following URD BMT can achieve a favorable long-term outcome and improved cognitive function. Future protocols must address the high risk of graft rejection or failure and the impact of GVHD in this patient population.

Bone Marrow Transplantation↗
PubMed1996

Pediatric large volume peripheral blood progenitor cell collections from patients under 25 kg: a primer.

Collection of peripheral blood progenitor cells from small pediatric patients provides many social and technical challenges not faced when collecting from adult patients. This paper provides a single institutions experience with 85 collections from 14 patients less than 25 kg of weight over a 2 year period. Specific challenges include obtaining venous access, anticoagulation, volume shifts, and obtaining patient cooperation. A systematic analysis of options for access, alternative modes of anticoagulation, and the effect of large ratios of extra-corporeal volume to patient's blood volume are discussed. Access uniformly required central venous catheters (CVC) ranging from 7-10 Fr. Anticoagulation included systemic heparinization titrating dose by activated clotting time in all cases and combined with citrate at a ratio of 1:25-1:30 in most cases. Collections were performed on a COBE Spectra, after priming with leukoreduced irradiated red cells and omitting both the initial 120cc diversion and rinse back of red cells at the end. Social challenges include issues of assent and ability to distract patients for the duration of a prolonged collection. Progenitor yields from collections from 14 patients were quantitated by CD34+ assay in all cases and CFU-GM in ten of 14 patients. A median of 4.5 x 10(6)/kg CD34+ cells were obtained for each collection. Complications, including those related to catheter access, are enumerated. In summary, large volume peripheral blood progenitor collection can be safely and efficaciously performed in small pediatric patients.

Body Weight↗
PubMed1996

Therapy of refractory or recurrent childhood acute myeloid leukemia using amsacrine and etoposide with or without azacitidine: a Pediatric Oncology Group randomized phase II study.

PURPOSE: A randomized study compared the combination of amsacrine (100 mg/m2/d on days 1 to 5) and etoposide (200 mg/m2/d on days 1 to 3) with the same two agents plus azacitidine (250 mg/m2/d on days 4 to 50) for the therapy of induction-resistant or relapse childhood acute myeloid leukemia (AML). PATIENTS AND METHODS: One hundred sixty-seven assessable children with AML who either had failed to respond to primary induction therapy (group 1, n = 41) or had relapsed (group 2, n = 126) were randomized. RESULTS: Overall, there were 56 complete responses (34%; SE 4%). Among primary refractory patients (group 1), the complete response rate was higher with the three-drug regimen (18% vs 53%, P = .03). In the relapsed patients (group 2), there was no difference in complete response rates related to treatment (31% vs 35%, P = .3). There were 17 early deaths. The major toxicities for both regimens were myelosuppression and infection. CONCLUSION: The overall complete response rate of 34% in this patient population is indicative of effective antileukemic activity. For patients with relapsed leukemia, the addition of azacitidine to etoposide and amsacrine did not improve response. The suggested advantage of the three-drug regimen for induction failures warrants further investigation.

Adolescent↗
PubMed1996

Contribution of a helix 5 locus to selectivity of hallucinogenic and nonhallucinogenic ligands for the human 5-hydroxytryptamine2A and 5-hydroxytryptamine2C receptors: direct and indirect effects on ligand affinity mediated by the same locus.

An important determinant of the neurobehavioral responses induced by a drug is its relative receptor selectivity. The molecular basis of ligand selectivity of hallucinogenic and nonhallucinogenic compounds of varying structural classes for the human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors was investigated with the use of reciprocal site-directed mutagenesis. Because these two closely related receptor subtypes differ in the amino acid present at position 5.46 (residues 242 and 222 in the sequences, respectively), the effects of corresponding substitutions in the 5-HT2A[S5.46(242)-->A] and 5-HT2C[A5.46(222)-->S] receptors were studied in tandem. By studying both receptors, the direct and indirect effects of mutations on affinity and selectivity can be distinguished. The ergolines studied, mesulergine (selective for the 5-HT2C receptor) and d-lysergic acid diethylamide (selective for the 5-HT2A receptor), reversed their relative affinity with mutations in each receptor, supporting a direct role of this locus in the selectivity of these ligands. However, interchange mutations in either receptor led to decreased or unchanged affinity for (+/-)-1-)(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and ketanserin, which have higher affinity for the 5-HT2A receptor, consistent with little contribution of this locus to the selectivity of these ligands. The indoleamines studied were affected differently by mutations in each receptor, suggesting that they bind differently to the two receptor subtypes. Mutation of this locus in the 5-HT2A receptor decreased the affinity of all indoleamines, whereas the interchange mutation of the 5-HT2C receptor did not affect indoleamine affinity. These results are consistent with a direct interaction between this side chain and indoleamines for the 5-HT2A receptor but not for the 5-HT2C receptor. Furthermore, this analysis shows that the higher affinity of 5-HT and tryptamine for the 5-HT2C receptor than for the 5-HT2A receptors is not due to the difference at this locus. The hallucinogens studied [d-lysergic acid diethylamide, psilocin, bufotenin, and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] fell into different classes in this analysis. For the classes of ligand studied, the side-chain difference at this position directly determines relative ligand selectivity only for ergolines and may contribute to the specific effects of hallucinogens in this class.

Amino Acid Sequence↗
PubMed1995

A locus of the gonadotropin-releasing hormone receptor that differentiates agonist and antagonist binding sites.

The decapeptide gonadotropin-releasing hormone controls reproductive function via interaction with a heptahelical G protein-coupled receptor. Because of molecular model of the receptor predicts that Lys121 in the third transmembrane helix contributes to the binding pocket, the function of this side chain was studied by site-directed mutagenesis. Substitution of Arg at this position preserved high affinity agonist binding, whereas Gln at this position reduced binding below the limits of detection. Leu and Asp at this locus abolished both binding and detectable signal transduction. The EC50 of concentration-response curves for coupling to phosphatidyl inositol hydrolysis obtained with the Gln121 receptor was more than 3 orders of magnitude higher than that obtained for the wild-type receptor. In order to determine whether the increased EC50 obtained with this mutant reflects an altered receptor affinity, the effect of decreases in wild-type receptor density on concentration-response curves was determined by irreversible antagonism. Progressively decreasing the concentration of the wild-type receptor increased the EC50 values obtained to a maximal level of 2.4 +/- 0.2 nM. Comparison of this value with the EC50 of 282 +/- 52 nM observed with the Gln121 receptor mutant indicates that the agonist affinity for this mutant is reduced more than 100-fold. In contrast, antagonist had comparable high affinities for the wild-type, Arg121, and Gln121 mutants. The results indicate that a charge-strengthened hydrogen bond donor is required at this locus for high affinity agonist binding but not for high affinity antagonist binding.

Amino Acid Sequence↗
PubMed1995

Related contribution of specific helix 2 and 7 residues to conformational activation of the serotonin 5-HT2A receptor.

A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT2A receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp-->Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn-->Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp-->Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.

Mutation↗
PubMed1995

Functional role of arginine-11 in the N-terminal helix of skeletal troponin C: combined mutagenesis and molecular dynamics investigation.

The two main structural differences between calmodulin (CaM) and skeletal troponin C (sTnC) are the absence in CaM of (i) the short N-terminal helix in TnC and (ii) the triplet KGK (residues 91-93; numbering according to chicken sTnC). It was recently shown that deletion of both structural groups from sTnC imparted to the resulting construct the CaM-like ability to activate phosphodiesterase (PDE) and to regulate force development in smooth muscle. To continue probing of the structural basis of the differential behavior of sTnC and CaM, residue Arg-11 in rabbit sTnC was mutated to Ala because the interactions of Arg-11 with distal residues in the N-terminal domain seem to link the N-terminal helix to the rest of the structure. The mutant exhibits CaM-like function in its ability to activate PDE (about 50% of CaM at 5 microM concentration). If, in addition, the KGK triplet is also deleted, PDE activation increases to about 80%. Both constructs retain their TnC function to nearly 100%. To explore the mechanistic basis of this remarkable observation, computational simulations of the molecular dynamics (MD) were carried out for both wild-type 4Ca2+.sTnC and the 4Ca2+.R11A mutant, and the results were compared to those from earlier simulations of 4Ca2+.CaM. Two types of structural changes observed from such simulations of the molecular dynamics of CaM had been considered to have a functional role: (i) a compaction to a more globular form and (ii) a reorientation of the Ca-binding domains around the central tether helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Electrostatic analysis of DNA binding properties in lysine to leucine mutants of TATA-box binding proteins.

The structures of the complexes between TATA-box binding proteins (TBPs) and DNA solved recently with X-ray crystallography identify both direct and indirect readout interactions. Examples of indirect readout mechanisms in these complexes are DNA bending and non-local electrostatic complementarity. An intriguing question arising from these structures is the role that a series of lysine residues may have in DNA binding. Thus, in the yeast complex, seven lysines are found to be close to the phosphate backbone, but they appear to form hydrogen bonds to the protein and not to be involved in any direct (or water-mediated) interactions with the DNA. The proposal based on the crystal structure, that these residues set up a delocalized electrostatic potential that stabilizes the complex with DNA, is evaluated here from calculations of the electrostatic potentials generated by the wild-type TBP and various lysine to leucine mutants. The results suggest a grouping of these mutants into three classes, based on their phenotypes and electrostatic profiles. As these groups are affected differently by specific measures taken to rescue DNA binding and transcription functions, the mechanistic inferences from the analysis can be probed experimentally in a manner that also reveals possible binding sites for transcription factors IIA and IIB to the TBP-DNA complex in the transcription preinitiation complex.

Base Sequence↗