Biomedical subjects

J N Heinrich

Publications and source records attributed to J N Heinrich.

6 recordsLinked to original sources

PubMed2000

Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae.

The Drosophila melanogaster ecdysone receptor (EcR) is produced in three isoforms, which mediate developmental processes such as metamorphosis. These isoforms were expressed in Saccharomyces cerevisiae to elucidate aspects of receptor transcription activity in a highly defined genetic model system. All three EcR isoforms showed ligand-independent transcriptional activation of an ecdysone reporter gene and the amount of activation correlated with the size of the N-terminal A/B (transactivation) domain present in the isoform: EcR-B1>EcR-A>>EcR-B2. Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR-B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B). Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR. Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N-terminus and the remainder from EcR (mAR¿EcR-CDEF) that was transcriptionally silent and inducible by Usp. In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR¿ EcR-CDEF. Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus. Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp. These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha. Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR.

Animals↗

PubMed1995

N51 competes 125I-interleukin (IL)-8 binding to IL-8R beta but not IL-8R alpha. Structure-function analysis using N51/IL-8 chimeric molecules.

We have demonstrated that the mouse chemokine N51, also known as KC, can compete for 125I-human interleukin-8 (IL-8) binding to NIH 3T3 cells expressing the human IL-8 receptor beta (NIH-IL-8R beta) but not the IL-8 receptor alpha (NIH-IL-8R alpha). In addition, we used the chimeras between N51 and IL-8 described previously (Heinrich, J. N., O'Rourke, E. C., Chen, L., Gray, H., Dorfman, K. S., and Bravo, R. (1994) Mol. Cell. Biol. 14, 2849-2861; Heinrich, J. N., and Bravo, R. (1995) J. Biol. Chem. 270, 4987-4989) to evaluate possible contributions of equivalent domains from each chemokine to binding and specificity. Specifically, the amino acid sequences between cysteines 2 and 3 or between cysteines 3 and 4 or the alpha-helical C-terminal end (domains I, II, and III, respectively) of one of the chemokines was exchanged with the corresponding sequence of the other and vice versa. Chimeras of IL-8 containing either domain II or III of N51 behaved similarly, but not identically, to IL-8 in competing 125I-IL-8 binding with both NIH-IL-8R alpha cells and NIH-IL-8R beta cells. The IL-8 chimera containing domain I of N51 did not compete. On the other hand, N51 competes 125I-IL-8 binding with NIH-IL-8R beta but not NIH-IL-8R alpha cells. The N51 chimera containing domain I of IL-8 was an agonist with NIH-IL-8R alpha cells and was an even more potent agonist with NIH-IL-8R beta cells. On the latter cells it was more potent than either IL-8 or N51. The N51 chimera containing domain II of IL-8, compared with N51, was a partial agonist with NIH-IL-8R alpha cells but was equivalent to N51 with NIH-IL-8R beta cells. However, N51 chimera containing domain III of IL-8 was a partial agonist with both cells. The results are consistent with the observations we originally made with human neutrophils and the NIH mouse IL-8R beta cells, i.e. domain I confers binding specificity for IL-8 and domains II and III of IL-8 and N51 may be interchangeable but they are not equivalent. Although we originally hypothesize that domains II and III confer binding specificity to N51, these results emphasize the role of domain III.

Amino Acid Sequence↗

PubMed1995

The orphan mouse receptor interleukin (IL)-8R beta binds N51. Structure-function analysis using N51/IL-8 chimeric molecules.

We have demonstrated that the orphan receptor representing the putative mouse (mu) homolog of the human (hu) interleukin-8 receptor beta (IL-8R beta) binds the mouse N51 cytokine, also known as KC. The muIL-8R beta gene was constitutively expressed in NIH 3T3 cells (NIH-muIL-8R beta). Cells and plasma membranes from the NIH-muIL-8R beta clone showed binding of 125I-N51 that was displaced by unlabeled N51. Other related cytokines were assayed for their ability to displace 125I-N51. MIP-2 and GRO alpha/MGSA competed as well as N51 for the receptor, but huIL-8 and NAP-2 did not compete at all. Chimeric molecules between IL-8 and N51 were used to extend the binding analysis. The segment between the conserved cysteines 2 and 3, named domain I; cysteines 3 and 4, domain II; and cysteine 4 and the C terminus, domain III of IL-8 were replaced by the corresponding domains of N51 and vice versa. When studying the binding of 125I-N51 and the hybrid molecules to the receptor, we observed that chimeras of N51 containing either domain I, II, or III of IL-8 were agonists of N51, and chimeras of IL-8 containing domain II or III of N51 were partial agonists of N51. These results demonstrate that domain I of N51 does not confer binding specificity and suggest that the region from the third cysteine to the C terminus of the N51 molecule is more important for binding to muIL-8R beta.

3T3 Cells↗

PubMed1994

Expression of the chemokine N51/KC in the thymus and epidermis of transgenic mice results in marked infiltration of a single class of inflammatory cells.

Transgenic mice expressing the chemokine N51/KC in thymus, skin, and tongue showed a marked infiltration of a single class of inflammatory cells (neutrophils) in the sites of transgene expression. In the thymus, neutrophils were most numerous in the cortex and juxta-medullary regions, often forming aggregates or clusters. A similar, but less intense, neutrophilic infiltrate occurred in close proximity to the epidermal basal layer of the tongue and skin. No morphologic evidence of injury was observed in the thymus, skin, or tongue of these transgenic mice, indicating that N51/KC expression induces recruitment but not inflammatory activation of neutrophils. The lack of activation in the thymus resulted in a large senescent neutrophilic population that was phagocytosed by thymic macrophages and epithelial-reticular cells. These results indicate that N51/KC is a neutrophil chemoattractant in vivo and establish these transgenic mice as effective models to study the phenomena of recruitment and clearance of neutrophils, events that are critical for the initiation and resolution of the inflammatory response.

Aging↗

PubMed1994

Biological activity of the growth factor-induced cytokine N51: structure-function analysis using N51/Interleukin-8 chimeric molecules.

The immediate-early gene N51/KC encodes a protein which following expression in the baculovirus system and purification to apparent homogeneity is able to induce chemotaxis and intracellular Ca2+ flux, to compete for 125I-labeled interleukin-8 (IL-8) binding, and upon iodination, to bind specifically to human neutrophils. The activity of N51/KC can be distinguished from that of IL-8 by a number of criteria. First, at equivalent concentrations, the specific binding of [125I]N51/KC to human neutrophils is about 10 times less than that of [125I]IL-8. Second, the competition studies of [125I]IL-8 with IL-8 define a single class of high-affinity receptors, while the presence of both a high- and a low-affinity class of receptors is defined by N51/KC. Third, although the changes in intracellular Ca2+ of fura-2/AM-preloaded human neutrophils elicited by N51/KC and IL-8 are similar, pretreatment of the cells with N51/KC did not result in a loss of response to a subsequent treatment with IL-8; in contrast, treatment with IL-8 did result in the subsequent desensitization to N51/KC. To further characterize N51/KC, mutants and hybrids of N51/KC and IL-8 were produced and analyzed for the ability to compete for [125I]IL-8 binding and elicit intracellular Ca2+ changes in human neutrophils. Two important observations came from these studies. First, the N51/IL-8I hybrid in which the N51/KC sequence between cysteines 2 and 3 (or first disulfide bond) is replaced by the corresponding sequence in IL-8 shows IL-8-like properties, indicating that this region is important for specific receptor recognition. Second, the N51 delta III and IL-8 delta III C-terminus deletion mutants were biologically inactive, but the hybrid molecules N51/IL-8III and IL-8/N51III, in which the C termini were exchanged, had biological activities similar to that of the wild-type molecules, demonstrating that the presence of the C terminus is essential for the biological activity of these chemokines but does not confer receptor specificity.

Amino Acid Sequence↗

PubMed1993

The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine.

We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.

Amino Acid Sequence↗