Receptor-specific Manner Research Articles

S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

BackgroundRecent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays.ResultsWe demonstrate that RSNOs, like S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors), inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors), but may only marginally affect signaling of others, such as adenosine A1, μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets), but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action.ConclusionThese in vitro studies show for the first time in a broader general context that RSNOs are capable of modulating GPCR signaling in a reversible and highly receptor-specific manner. Given that the enzymatic machinery responsible for endogenous NO production is located in close proximity with the GPCR signaling complex, especially with that for several receptors whose signaling is shown here to be modulated by exogenous RSNOs, our data suggest that GPCR signaling in vivo is likely to be subject to substantial, and highly receptor-specific modulation by NO-derived RSNOs.

Dual mode regulation of migration by lysophosphatidic acid in human gastric cancer cells

Lysophosphatidic acid (LPA), which interacts with at least three G protein-coupled receptors (GPCRs), LPA1/Edg-2, LPA2/Edg-4, and LPA3/Edg-7, is a lipid mediator with diverse effects on various cells. Here, we investigated the expression profiles of LPA receptors and patterns of LPA-induced migration in gastric cancer cells. Northern blot analysis revealed that various gastric cancer cells expressed variable levels of LPA1, LPA2, and LPA3 without a consistent pattern. Using a Boyden chamber assay, LPA markedly increased cell migration of LPA1-expressing cells, the effects of which were almost totally abrogated by Ki16425, an LPA antagonist against LPA1 and LPA3. In contrast, LPA by itself did not significantly induce migration in MKN28 and MKN74 cells, which exclusively expressed LPA2. However, when hepatocyte growth factor (HGF) was placed with LPA in the lower chamber, LPA induced migration of these cells in a dose-dependent manner. Immunoprecipitation analysis revealed that LPA induced transient tyrosine phosphorylation of c-Met in LPA2-expressing cells, which suggests that the transactivation of c-Met by LPA causes a cooperative migratory response with HGF to these cells. Our results indicate that LPA regulates the migration of gastric cancer cells in a receptor-specific manner and suggest that the expression pattern of LPA receptors may affect the metastatic behavior of gastric cancer.

DAXX, FLASH, and FAF-1 modulate mineralocorticoid and glucocorticoid receptor-mediated transcription in hippocampal cells--toward a basis for the opposite actions elicited by two nuclear receptors?

Mineralocorticoid (MR) and glucocorticoid (GR) receptors are two closely-related members of the steroid nuclear receptor family of transcription factors that bind common ligands in the brain (corticosterone and cortisol) and supposedly have identical hormone response elements. This raises the important question of how they can elicit differential biological actions in neurons in which they are often colocalized. One plausible explanation is that they differentially recruit proteins (coregulators or other receptor-interacting factors) through cell-specific interactions with regions that diverge between MR and GR to modulate target gene transcription in a receptor-specific manner. We therefore performed a yeast-two-hybrid screening of a human brain cDNA library with an AF1-containing region of the human MR as bait. This screening revealed several potential MR-interacting partners; among them were several clones bearing homology to DAXX, FLASH, and FAF-1, all previously implicated in apoptosis. Coexpression of candidate clones in a mouse hippocampal cell line confirmed these interactions in a mammalian neural cell environment as well. In transient transactivation assays, DAXX and FLASH influenced MR- and GR-driven transcription of the MMTV-Luc reporter similarly; in contrast, although FAF-1 did not transactivate GR, it did selectively stimulate MR-mediated transcription. Thus, the present findings, that 1) DAXX, FLASH, and FAF-1 modulate the transcriptional activities of MR and GR and that 2) FAF-1 selectively coactivates only MR, provide possible clues for how these closely related receptors might differentially influence neuronal function.

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation.

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

In vitro selection of viral vectors with modified tropism: the adeno-associated virus display

Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.

PPARs of the Heart

The peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated transcription factors within the broad nuclear receptor superfamily. The PPAR family includes three members encoded by distinct genes: α, β/δ, and γ (see reviews1,2). PPARα was originally identified as the intracellular receptor for a class of nongenotoxic rodent hepatocarcinogens, which includes the hypolipidemic drug clofibrate, a potent inducer of hepatic peroxisomal proliferation and hypolipidemic agent. The three PPARs are now distinguished by tissue- and developmental-specific patterns of expression and by the distinct, albeit overlapping, nature of ligands capable of activating each receptor. PPARα, which is abundant in tissues with high rates of mitochondrial fatty acid oxidation, such as heart, liver, and kidney, regulates a wide variety of target genes involved in cellular lipid catabolism. In contrast, PPARγ, an adipose-enriched nuclear receptor, directs the expression of genes involved in adipocyte differentiation and fat storage. The function of the ubiquitously expressed PPARβ/δ, is not well understood although some evidence suggests that it exerts actions on the epidermis and activates antiinflammatory programs. Ligand activation of PPARs leads to obligate heterodimerization with the 9- cis retinoic acid-activated receptor, RXR, promoting binding of the complex to cognate DNA response elements within PPAR target gene promoter regions (Figure). A variety of natural and synthetic compounds including fatty acids, eicosanoids, and arachidonic acid derivatives can serve as activators of the PPARs, some in a receptor-specific manner. However, the true endogenous ligands have not been identified. PPAR transcriptional regulatory complex. PPARs bind to sequence-specific target elements (PPREs) as a liganded heterodimer with the retinoid X receptor (RXR). Major functions based on known target genes and examples of relevant tissues (in parentheses) for each member of the PPAR family are shown. The question mark indicates that target genes for PPARβ/δ are largely unidentified. PPARα has been shown to …

Cyclin D3 Is a Cofactor of Retinoic Acid Receptors, Modulating Their Activity in the Presence of Cellular Retinoic Acid-binding Protein II

Ligand-induced transcription activation of retinoic acid (RA) target genes by nuclear receptors (retinoic acid (RAR) and retinoid X (RXR) receptors) depends on the recruitment of coactivators. We have previously demonstrated that the small 15-kDa cellular RA-binding protein II (CRABPII) is a coactivator present in the RA-dependent nuclear complex. As identifying cell-specific partners of CRABPII might help to understand the novel control of RA signaling, we performed a yeast two-hybrid screen of a hematopoietic HL-60 cDNA library using human CRABPII as bait and have subsequently identified human cyclin D3 as a partner of CRABPII. Cyclin D3 interacted with CRABPII in a ligand-independent manner and equally bound RAR alpha, but not RXR alpha, and only in the presence of RA. We further show that cyclin D3 positively modulated RA-mediated transcription through CRABPII. Therefore, cyclin D3 may be part of a ternary complex with CRABPII and RAR. Finally, we show that cyclin D3 expression paralleled HL-60 differentiation and arrest of cell growth. These findings led us to speculate that control of cell proliferation during induction of differentiation may directly involve, at the transcriptional level, nuclear receptors, coactivators, and proteins of the cell cycle in a cell- and nuclear receptor-specific manner.

Purinergic P2X(2) receptor desensitization depends on coupling between ectodomain and C-terminal domain.

The wild-type P2X(2) purinergic receptor (P2X(2a)R) and its splice form lacking the intracellular Val(370)-Gln(438) C-terminal sequence (P2X(2b)R) respond to ATP stimulation with comparable EC(50) values and peak current/calcium responses but desensitize in a receptor-specific manner. P2X(2a)R desensitizes slowly and P2X(2b)R desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X(2a)R and P2X(2b)R. Both receptors showed similar EC(50) values (estimated from the peak calcium response) and IC(50) values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP <or= ATP <or= ATPgammaS < BzATP << alphabeta-meATP, and the IC(50) values for agonists were shifted to the right compared with their EC(50) values. Furthermore, the ATP-induced receptor-subtype specific pattern of desensitization was mimicked by high- but not by low-efficacy agonists, suggesting a ligand-specific desensitization pattern. To test this hypothesis, we generated chimeric P2X(2a)R and P2X(2b)R containing the Val(60)-Phe(301) ectodomain sequence of P2X(3)R and Val(61)-Phe(313) ectodomain sequence of P2X(7)R instead the native Ile(66)-Tyr(310) sequence. The mutated P2X(2a)+X(3)R and P2X(2b)+X(3)R exhibited comparable EC(50) values for ATP, BzATP, and alphabeta-meATP in the submicromolar concentration range and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X(2)+X(7)R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. These results suggest that efficacy of agonists for the ligand-binding domain of P2X(2)Rs reflects the strength of desensitization controlled by their C-terminal structures.

Evolution of discrete Notch-like receptors from a distant gene duplication in Caenorhabditis.

SUMMARY Caenorhabditis elegans possesses two Notch-like receptors, LIN-12 and GLP-1, which have both overlapping and individual biological functions. We examined the lin-12 and glp-1 genes in closely related nematodes to learn about their evolution. Here we report molecular and functional analyses of lin-12 orthologs from two related nematodes, C. briggsae (Cb) and C. remanei (Cr). In addition, we compare these lin-12 findings with similar studies of Cb-glp-1 and Cr-glp-1 orthologs. Cb-LIN-12 and Cr-LIN-12 retain the same number and order of motifs as Ce-LIN-12. Intriguingly, we find that LIN-12 conservation differs from that of GLP-1 in two respects. First, individual motifs are conserved to a different degree for the two receptors. For example, the transmembrane domain is 16-32% identical among LIN-12 orthologs but 65-70% identical among GLP-1 orthologs. Second, certain amino acids are conserved in a receptor-specific manner, a phenomenon most prevalent in the CC-linker. We suggest that LIN-12 and GLP-1 have been molded by selective constraints that are receptor specific and that the two proteins may not be entirely interchangeable. To analyze the functions of the lin-12 orthologs, we used RNA-mediated interference (RNAi). Cb-lin-12(RNAi) or Cr-lin-12(RNAi) progeny are nearly 100% Lag, a larval lethality typical of C. elegans lin-12 glp-1 double mutants, but not the primary defect observed in Ce-lin-12 null mutants or Ce-lin-12(RNAi). Therefore, LIN-12 functions are similar, but not identical, among the Caenorhabditis species. We suggest that ancestral functions may have been divided between LIN-12 and GLP-1 receptors in a process contributing to the retention of both genes after gene duplication (i.e., subfunctionalization).

Receptor-specific regulation of phosphatidylinositol 3'-kinase activation by the protein tyrosine phosphatase Shp2.

Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological systems. Many RTKs transmit similar signals, raising questions about how specificity is achieved. One potential mechanism for RTK specificity is control of the magnitude and kinetics of activation of downstream pathways. We have found that the protein tyrosine phosphatase Shp2 regulates the strength and duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit increased association of the p85 subunit of PI3K with the scaffolding adapter Gab1 compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells reconstituted with WT Shp2. Far-Western analysis suggests increased phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant cells following EGF stimulation. Analogous results are obtained in fibroblasts inducibly expressing dominant-negative Shp2. Our results suggest that, in addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner.

Kupffer cell-mediated recruitment of rat dendritic cells to the liver: Roles of N-Acetylgalactosamine-Specific sugar receptors

Tissue recruitment of dendritic cells (DCs) is essential for antigen presentation. This study aimed to examine cellular and molecular mechanisms for DC recruitment to the liver. Purified rat DCs were injected into circulation and their traffics were analyzed in normal and Kupffer cell-depleted rats by intravital confocal microscopy and immunohistology. Affinities of DCs to sinusoidal cells were examined by a cell-binding assay. DC precursor recruitment was induced by particulate injection. Both DC precursors and DCs at the antigen-transporting stage could be recruited to the liver, and their majority initially showed a selective binding to Kupffer cells. In the Kupffer cell-depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides showed that sugar residues consisting of N-acetylgalactosamine were necessary for this binding. The binding was calcium-dependent, implying the C-type lectin involvement. Furthermore, DCs could endocytose N-acetylgalactosamine polymers in a receptor-specific manner. The DC-Kupffer cell binding through N-acetylgalactosamine-specific C-type lectin-like receptors is crucial for DC recruitment to the liver. Rat DCs at least partly possess receptors for endocytosis of galactosylated antigens. These DC receptors as well as Kupffer cell lectins are presumably responsible for this binding.

Nonviral gene delivery to human breast cancer cells by targeted Ad5 penton proteins.

The capsid proteins of adenovirus serotype 5 (Ad5) are key to the virus' highly efficient cell binding and entry mechanism. In particular, the penton base plays a significant role in both viral internalization and endosome penetration. We have produced an adenovirus penton fusion protein (HerPBK10) containing moieties for DNA transport and targeted delivery to breast cancer cells. HerPBK10 binds DNA through a polylysine appendage, while the EGF-like domain of the heregulin-alpha(1) isoform is used as the targeting ligand. This ligand binds with high affinity to HER2/3 or HER2/4 heterodimers, which are overexpressed on certain aggressive breast cancers. In addition, this ligand is rapidly internalized after binding, thus adding to the utility of heregulin for targeting. HerPBK10 binds MDA-MB-453 breast cancer cells in a receptor-specific manner, and mediates the entry of a reporter plasmid in MDA-MB-453 cells in culture. Delivery can be competed by excess heregulin peptide, thus confirming receptor specificity. Importantly, the penton segment appears to contribute significantly to enhanced delivery. Complexes containing HerPBK10 and DNA have been optimized to provide targeted gene delivery to breast cancer cells in vitro. We demonstrate that delivery can be accomplished in the presence of serum, thus suggesting a potential use for in vivo delivery.

Polarized expression of G protein-coupled receptors and an all-or-none discharge of Ca2+ pools at initiation sites of [Ca2+]i waves in polarized exocrine cells.

In the present work we examined localization and behavior of G protein-coupled receptors (GPCR) in polarized exocrine cells to address the questions of how luminal to basal Ca(2+) waves can be generated in a receptor-specific manner and whether quantal Ca(2+) release reflects partial release from a continuous pool or an all-or-none release from a compartmentalized pool. Immunolocalization revealed that expression of GPCRs in polarized cells is not uniform, with high levels of GPCR expression at or near the tight junctions. Measurement of phospholipase Cbeta activity and receptor-dependent recruitment and trapping of the box domain of RGS4 in GPCRs complexes indicated autonomous functioning of G(q)-coupled receptors in acinar cells. These findings explain the generation of receptor-specific Ca(2+) waves and why the waves are always initiated at the apical pole. The initiation site of Ca(2+) wave at the apical pole and the pattern of wave propagation were independent of inositol 1,4,5-trisphosphate concentration. Furthermore, a second Ca(2+) wave with the same initiation site and pattern was launched by inhibition of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps of cells continuously stimulated with sub-maximal agonist concentration. By contrast, rapid sequential application of sub-maximal and maximal agonist concentrations to the same cell triggered Ca(2+) waves with different initiation sites. These findings indicate that signaling specificity in pancreatic acinar cells is aided by polarized expression and autonomous functioning of GPCRs and that quantal Ca(2+) release is not due to a partial Ca(2+) release from a continuous pool, but rather, it is due to an all-or-none Ca(2+) release from a compartmentalized Ca(2+) pool.

Intracellular signalling. Receptor-specific messenger oscillations.

The cytosolic molecule inositol-1,4,5-trisphosphate (InsP3) acts as a messenger to link receptors at the cell surface with alterations in calcium concentration inside the cell1, but it is not clear how production of InsP3 is related to the often-complex calcium response2. Here we use a fluorescent biosensor to visualize InsP3 synthesis in individual cells in real time and show that this is periodically switched on and off in a receptor-specific manner. Our findings are consistent with intracellular calcium oscillations being generated by either fluctuating or sustained concentrations of InsP3, which may allow diversity of signalling through the same signal-transduction pathway.

Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin.

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen.

Steroid receptors mediate responses to lipophilic hormones in a tissue- and ligand-specific manner. To identify nonreceptor proteins that confer specificity or regulate steroid signaling, we screened a human cDNA library in a steroid-responsive yeast strain. One of the identified cDNAs, isolated in the screen as ligand effect modulator 6, showed no homology to yeast or Caenorhabditis elegans proteins but high similarity to the recently described mouse coactivator PGC-1 and was accordingly termed hPGC-1. The hPGC-1 DNA encodes a nuclear protein that is expressed in a tissue-specific manner and carries novel motifs for transcriptional regulators. The expression of hPGC-1 in mammalian cells enhanced potently the transcriptional response to several steroids in a receptor-specific manner. hPGC-1-mediated enhancement required the receptor hormone-binding domain and was dependent on agonist ligands. Functional analysis of hPGC-1 revealed two domains that interact with steroid receptors in a hormone-dependent manner, a potent transcriptional activation function, and a putative dimerization domain. Our findings suggest a regulatory function for hPGC-1 as a tissue-specific coactivator for a subset of nuclear receptors.

Differential effects of interferon-γ on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fcγ receptors

AbstractThe objectives were to determine whether there are differences in the mechanisms of lipoprotein metabolism associated with different FcγRs and how metabolism associated with FcγRs compares to that mediated by scavenger receptors (SRA). To analyze lipoprotein metabolism in a receptor-specific manner, bispecific antibodies were used to target low density lipoproteins (LDL) labeled with 125I or [3H]cholesterol linoleate to FcγRI or FcγRIIA in human macrophages. Interferon-γ (IFN-γ), which stimulates expression of FcγRI while inhibiting expression of SRA, was used to help delineate differences in metabolism between each receptor. For each receptor, the total amount of lipoprotein degradation paralleled changes in receptor expression induced by IFN-γ. In particular, while SRA-mediated degradation typically exceeded degradation mediated by FcγRI, in IFN-γ-treated cells degradation associated with FcγRI and SRA was similar. Assay of [3H]cholesterol linoleate-labeled lipoproteins indicated that total uptake and hydrolysis of [3H]cholesterol linoleate was similar for each class of receptor, and inhibited by IFN-γ. For FcγRI versus FcγRIIA, in the presence or absence of IFN-γ, the [3H]cholesterol derived from FcγRIIA-mediated uptake was preferentially targeted for re-esterification to [3H]cholesterol oleate, in comparison to that resulting from hydrolysis of [3H]cholesterol linoleate incorporated by selective uptake. For SRA, the formation of [3H]cholesterol oleate, which was substantial in control cells, was significantly inhibited in the presence of IFN-γ. We conclude that there may be differences in cholesterol trafficking with respect to lipoprotein immune complex metabolism mediated by different classes of FcγRs. —Morganelli, P. M., S. M. Kennedy, and T. I. Mitchell. Differential effects of interferon-γ on metabolism of lipoprotein immune complexes mediated by specific human macrophage Fcγ receptors. J. Lipid Res. 2000. 41: 405–415.

Alpha-Thrombin stimulates contraction of human bronchial rings by activation of protease-activated receptors.

In a variety of diseases, inflammation causes microvascular leakage and activates thrombin. Evidence suggests that thrombin increases cytosolic calcium and stimulates human airway smooth muscle (ASM) cell proliferation. The receptor subtypes, however, that mediate the effects of thrombin on ASM cell growth or calcium mobilization remain unknown. In this study, we postulate that thrombin, which activates specific protease-activated receptors (PARs), also stimulates contraction of isolated human bronchial rings. With the use of intact human bronchial rings, alpha-thrombin (1-20 U/ml) increased bronchial tone to 19 +/- 3% of basal tone (P = 0.008; n = 5 experiments) and represents 20 +/- 8% of the maximum carbachol response. The EC(50) for thrombin-induced force generation was 12.2 U/ml (95% confidence interval 9.9-15.3 U/ml) and was not altered in bronchial rings that had the epithelium removed. In parallel experiments, a specific thrombin receptor-activating peptide (TRAP-14; 0.1-100 micromol/l) increased isometric tension to levels (14 +/- 2%; P = 0.0005; n = 5 experiments) comparable to those rings stimulated with thrombin. To characterize the receptors that mediate thrombin effects on human ASM, the expression of PARs in cultured human ASM cells was analyzed by RT-PCR analysis with specific primers for PARs. In these cells, PAR1 (thrombin receptor), PAR2, and PAR3 were expressed at comparable levels. In other experiments using immunocytochemical staining with specific antibodies to PAR1 and PAR2, we showed that ASM in bronchial rings and cultured ASM cells express PAR1 and PAR2 proteins. Taken together, these studies suggest that alpha-thrombin, in a receptor-specific and dose-dependent manner, induces contraction of bronchial rings in vitro. In addition, cultured human ASM cells express mRNA of PAR1, PAR2, and PAR3 and express PAR1 and PAR2 protein. Further studies are needed to determine whether alpha-thrombin plays a role in stimulating bronchoconstriction in inflammatory airway diseases such as asthma and bronchiolitis obliterans.

A putative alpha-helical G beta gamma-coupling domain in the second intracellular loop of the 5-HT1A receptor.

We have identified a conserved threonine residue in the second intracellular (i2) loop of the 5-HT1A receptor that when mutated to alanine prevents coupling to G beta gamma-mediated signaling, while preserving G alpha i-induced actions. In this review, we investigate the characteristics and potential role of the i2 domain in the coupling of the 5-HT1A receptor and other receptors to G proteins. The i2 domain, as well as portions of the i3 domain, is predicted to form an amphipathic alpha-helix with a positively charged face and a hydrophobic face. Mutagenesis experiments support a model in which the hydrophobic faces of these alpha-helical domains form an intracellular binding "pocket" for interaction with G proteins. Embedded in the hydrophobic face, Thr 149 is crucial for signaling through G beta gamma subunits, perhaps via interaction with its hydroxyl side-chain. Mutation of other residues of the i2 domain of Gi-coupled receptors is required to substantiate the importance of the alpha-helical i2 domain in receptor-G beta gamma signaling. If confirmed in other receptors, these results support a general model in which activated receptor and G beta gamma subunits remain associated to interact with effectors in a receptor-specific manner.

Targeting Dendrimer-Chelates to Tumors and Tumor Cells Expressing the High-Affinity Folate Receptor

The authors developed a new method for delivering contrast agents to tumors and tumor cells. Gadolinium complexes of folate-conjugated dendrimer-chelates increased the longitudinal relaxation rate of tumor cells expressing the high-affinity folate receptor, hFR. The coupling of folate to polymeric chelates, composed of a dendrimer backbone, targets these chelates to endogenous folate binding proteins. These proteins exist in both the serum of patients with cancer and on the cell surface of many human cancers of epithelial origin. The authors attached folic acid to a generation four ammonia core polyamidoamine dendrimer. The folate-dendrimer was reacted with 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid to form the polymeric chelate f-PAMAM-TU-DTPA. For fluorescent studies, the generation four dendrimer was reacted with fluorescein-5-isothiocyanate and carboxytetramethylrhodamine succinimidyl ester, followed by capping the remaining amines with succinic anhydride. The study results show that cells accumulate the folate-conjugated dendrimer in a receptor specific manner. Tumor cells expressing the high-affinity folate receptor showed a 650% increase in the mean fluorescence. This increase occurred with a rapid rise to 325%, followed by a slow increase to 650%. It required both the expression of the hFR and the coupling of folic acid to the dendrimer. Excess free folic acid inhibited the binding of the folate conjugated polymer. Fluorescent microscopic study showed that the folate-conjugated dendrimer binds to the cell surface and is accumulated within the cells. Treatment of tumor cells that express the hFR with gadolinium complexes of the folate-conjugated polymeric chelate increases the longitudinal relaxation rate by 110%. This increase was inhibited by an excess of free folic acid. These data demonstrate that folate-conjugated magnetic resonance imaging contrast agents represent a promising new approach to tumor targeting.