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Biomedical subjects

T W Schwartz

Publications and source records attributed to T W Schwartz.

At least 19 recordsLinked to original sources
PubMed1997

Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time approximately 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1-mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

Animals↗
PubMed1997

A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

Amino Acid Sequence↗
PubMed1997

Characterization of non-peptide antagonist and peptide agonist binding sites of the NK1 receptor with fluorescent ligands.

Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the Nalpha position were synthesized and characterized. Peptides modified at the alpha-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18, 000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyl-labeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the non-peptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.

Animals↗
PubMed1997

Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist.

The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.

Animals↗
PubMed1997

[7TM receptors--from the olfactory system and new hormones against HIV infection].

It has recently become evident that all types of chemical messengers, hormones and transmitters act through membrane receptors which constitute our largest superfamily of proteins, i.e. the G protein-coupled receptors. These proteins, which are characterized by having seven transmembrane segments (7TM), also act as, for example sensors for light and odor components in our sensory systems. Already today monoamine 7TM receptors are the target for many drugs; however, the development of non-peptide ligands for a variety of peptide receptors indicates that probably all 7TM receptors can become pharmacotherapeutic targets. The discovery that chemokine receptors function as the crucial cofactors for cell entry of HIV-1 suggests that antagonists or agonists for one or more chemokine 7TM receptor could be interesting agents against AIDS. The occurrence of a multitude of orphan 7TM receptors without known ligand indicates, that surprisingly large areas within endocrinology and neuroscience are still today waiting to be characterized.

GTP-Binding Proteins↗
PubMed1997

Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors.

G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.

Binding Sites↗
PubMed1997

Dual agonistic and antagonistic property of nonpeptide angiotensin AT1 ligands: susceptibility to receptor mutations.

Two nonpeptide ligands that differ chemically by only a single methyl group but have agonistic (L-162,782) and antagonistic (L-162,389) properties in vivo were characterized on the cloned angiotensin AT1 receptor. Both compounds bound with high affinity (K(I) = 8 and 28 nM, respectively) to the AT1 receptor expressed transiently in COS-7 cells as determined in radioligand competition assays. L-162,782 acted as a powerful partial agonist, stimulating phosphatidylinositol turnover with a bell-shaped dose-response curve to 64% of the maximal level reached in response to angiotensin II. Surprisingly, L-162,389 also stimulated phosphatidylinositol turnover, albeit only to a small percentage of the angiotensin response. The prototype nonpeptide AT1 agonist L-162,313 gave a response of approximately 50%. The apparent EC50 values for all three compounds in stimulating phosphatidylinositol turnover were similar, approximately 30 nM, corresponding to their binding affinity. Each of the three compounds also acted as angiotensin antagonists, yet in this capacity the compounds differed markedly, with IC50 values ranging from 1.05 x 10(-7) M for L-162,389 to 6.5 x 10(-6) for L-162,782. A series of point mutations in the transmembrane segments (TMs) of the AT1 receptor had only minor effect on the binding affinity of the nonpeptide compounds, with the exception of A104V at the top of TM III, which selectively impaired the binding of L-162,782 and L-162,389. Substitutions in the middle of TM III, VI, or VII, which did not affect the binding affinity of the compounds, impaired or eliminated the agonistic efficacy of the nonpeptides but with only minor or no effect on the angiotensin potency or efficacy. Thus, in the N295D rat AT1 construct, L-162,782, L-162,313, and L-162,389 all antagonized the angiotensin-induced phosphatidylinositol turnover with surprisingly similar IC50 values (90-180 nM), and they all bound with unaltered, high affinity (22-36 nM). However, L-162,313 and L-162,782 could stimulate phosphatidylinositol turnover to only 20% of that of angiotensin. It is concluded that minor chemical modifications of either the compound or the receptor can dramatically alter the agonistic efficacy of biphenyl imidazole compounds on the AT1 receptor without affecting their affinity, as determined in binding assays, and that a number of substitutions in the middle of the TM segments affect the efficacy of nonpeptide agonists as opposed to angiotensin.

Angiotensin II↗
PubMed1996

Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

Amino Acid Sequence↗
PubMed1996

Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering.

A high affinity, tridentate metal ion site has been constructed previously by His substitutions in an antagonist binding site located between transmembrane segment (TM)-V and TM-VI in the substance P NK-1 receptor. Here, an attempt is made to probe helix-helix interactions systematically in the NK-1 receptor by engineering of bis-His Zn(II) sites. His residues were introduced at selected positions individually and in combinations in the exterior segments of TM-II, III and V in both the wild-type background and after Ala substitution of naturally occurring His residues, and the increase in the affinity for Zn(II) was monitored in competition binding experiments with iodinated substance P or a tritiated non-peptide antagonist. In this way, two high affinity bis-His sites were constructed between position 193 in TM-V (Glu193, G1uV:01) and position 109 in TM-III (Asn1O9, AsnIII:05) as well as between the neighboring, naturally occurring His108 in TM-III (HisIII:04) and position 92 in TM-II (Tyr92, TyrII:24), respectively. Functionally, the coordination of zinc ions at these two sites blocked the receptor as it antagonized the substance P-induced increase in phosphatidylinositol turnover. It is concluded that the bis-His zinc sites from the central TM-III helix to TM-II and -V, respectively, together with the interconnected, previously constructed tridentate site between TM-V and -VI, constitute a basic network of distance constraints for the molecular models of receptors with seven transmembrane segments which, for example, strongly support an anti-clockwise orientation of the seven helical bundle as viewed from the extracellular space.

Amino Acid Sequence↗
PubMed1996

Construction of a high affinity zinc switch in the kappa-opioid receptor.

Very limited structural information is available concerning the superfamily of G-protein-coupled receptors with their seven-transmembrane segments. Recently a non-peptide antagonist site was structurally and functionally replaced by a metal ion site in the tachykinin NK-1 receptor. Here, this Zn(II) site is transferred to the kappa-opioid receptor by substituting two residues at the outer portion of transmembrane V (TM-V), Asp223 and Lys227, and one residue at the top of TM-VI, Ala298, with histidyl residues. The histidyl residues had no direct effect on the binding of either the non-peptide antagonist [3H]diprenorphine or the non-peptide agonist, [3H]CI977, just as these mutations/substitutions did not affect the apparent affinity of a series of other peptide and non-peptide ligands when tested in competition binding experiments. However, zinc ions in a dose-dependent manner prevented binding of both agonist and antagonist ligands with an apparent affinity for the metal ion, which gradually was built up to 10(-6) M. This represents an increase in affinity for the metal ion of about 1000-fold as compared with the wild-type kappa receptor and is specific for Zn(II) as the affinity for e.g. Cu(II) was almost unaffected. The direct transfer of this high affinity metal ion switch between two only distantly related receptors indicates a common overall arrangement of the seven-helix bundle among receptors of the rhodopsin family.

Amino Acid Sequence↗
PubMed1996

Biosynthesis of neuropeptide Y in porcine tissues and generation of N-terminal fragments in neuroblastoma cell lines.

The biosynthesis of neuropeptide Y (NPY) was investigated to determine the efficiency of synthesis and processing of the precursor. In brain tissues examined, the major product was amidated NPY(1-36). Although this was also the major product in adrenal and heart atrium, a minor portion of the immunoreactivity was identified as unprocessed precursor. NPY degradation was investigated using SK-N-MC and SMS-MSN cells in conjunction with iodinated NPY tracers, labeled in either the tyrosine-1 or the tyrosine-36 position. Similar patterns of degradation were observed with both cell lines, and it would appear that the initial proteolytic attack on NPY(1-36) generates predominately N-terminal fragments.

Adrenal Glands↗
PubMed1996

Radioligand-dependent discrepancy in agonist affinities enhanced by mutations in the kappa-opioid receptor.

A series of kappa/mu receptor chimeras and a number of kappa receptors substituted in the second transmembrane segment (TM-II) were investigated using as radioligands, respectively, the kappa-selective agonist [3H]C1977 and the nonselective opioid antagonist [3H]diprenorphine (DIP). All of the receptor constructs bound [3H]DIP with similar and high affinity, whereas the apparent affinity of the nonpeptide agonist C1977, when estimated in competition binding with the antagonist [3H]DIP, was impaired between 42- and > 500-fold in the kappa/mu chimeras and between 64- and 153-fold in three of the kappa receptor mutants that had been substituted in the TM-II segment. However, homologous competition binding experiments, using [3H]C1977 as radioligand, showed that the high affinity binding of this nonpeptide agonist was in fact not impaired in four of the kappa/mu chimeras and in three TM-II substituted kappa receptors compared with the wild-type kappa receptor. In all cases in which mutations decreased the apparent affinity of C1977 without affecting its actual affinity, as determined in homologous assays using [3H]C1977, the calculated number of receptor sites (Bmax) was decreased. In three of the kappa/mu constructs, binding of [3H]C1977 was undetectable, indicating that in these chimeras the affinity of the nonpeptide agonist had actually been affected. Also, for the kappa-selective peptide agonist dynorphin A(1-8), the measured affinity for the receptor mutants was strongly dependent on whether it was determined using the antagonist [3H]DIP or the agonist [3H]C1977 in that < or = 800-fold higher Ki values were determined in competition with the antagonist. It is concluded that mutations in the kappa-opioid receptor can cause large discrepancies between the affinity determined for agonists in homologous versus heterologous competition binding assays and that this pattern, which is compatible with a partial uncoupling of receptors, is observed in surprisingly many types of receptor mutations.

Animals↗