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Biomedical subjects

M A Goldsmith

Publications and source records attributed to M A Goldsmith.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of a variable region within the cytoplasmic tail of the IL-2 receptor beta chain that is required for growth signal transduction.

Interleukin-2 (IL-2) regulates numerous biological events, including T lymphocyte proliferation. Interleukin-2 receptor (IL-2R)-mediated signaling is triggered by ligand-induced heterodimerization of the IL-2R beta and gamma c subunits, which results in the activation of signaling intermediates that are associated with either IL-2R beta or gamma c. Previous mutagenesis studies of the IL-2R beta cytoplasmic tail demonstrated that the partially conserved box 1 and box 2 motifs and specific tyrosine residues are critical for growth signaling. By deletion and alanine scanning mutagenesis, another set of residues that are critical for IL-2R-mediated signaling has now been identified. These residues lie within the divergent 35-amino acid "spacer" region separating box 1 and box 2. The role of this receptor subregion in early phases of IL-2R signaling was evaluated using BA/F3 stable cell lines expressing three functionally impaired mutants from this region. All three cell lines displayed substantially diminished growth responsiveness to IL-2. Receptor-mediated STAT factor activation, IL-2R beta phosphorylation, and Janus kinase activation were also markedly impaired. These findings indicate that this variable spacer region, which we have termed the V-box, is essential for the initiation of IL-2R-mediated signal transduction.

Amino Acid Sequence↗
PubMed1995

Growth signal transduction by the human interleukin-2 receptor requires cytoplasmic tyrosines of the beta chain and non-tyrosine residues of the gamma c chain.

To evaluate the possible role for receptor-based tyrosine phosphorylation in growth signaling induced by interleukin-2 (IL-2), a series of substitution tyrosine mutants of the IL-2 receptor beta and gamma c chains was prepared and analyzed. Concurrent mutation of all six of the cytoplasmic tyrosines present in the beta chain markedly inhibited IL-2-induced growth signaling in both pro-B and T cell lines. Growth signaling in a pro-B cell line was substantially reconstituted when either of the two distal tyrosines (Tyr-392, Tyr-510) was selectively restored in the tyrosine-negative beta mutant, whereas reconstitution of the proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function. Furthermore, at least one of the two cytoplasmic tyrosines that is required for beta chain function was found to serve as a phosphate acceptor site upon induction with IL-2. Studies employing a chimeric receptor system revealed that tyrosine residues of the beta chain likewise were important for growth signaling in T cells. In contrast, although the gamma c subunits is a target for tyrosine phosphorylation in vivo, concurrent substitution of all four cytoplasmic tyrosines of this chain produced no significant effect on growth signaling by chimeric IL-2 receptors. However, deletion of either the Box 1, Box 2, or intervening (V-Box) regions of gamma c abrogated receptor function. Therefore, tyrosine residues of beta but not of gamma c appear to play a pivotal role in regulating growth signal transduction through the IL-2 receptor, either by influencing cytoplasmic domain folding or by serving as sites for phosphorylation and subsequent association with signaling intermediates. These findings thus highlight a fundamental difference in the structural requirements for IL-2R beta and gamma c in receptor-mediated signal transduction.

Amino Acid Sequence↗
PubMed1995

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

Amino Acid Sequence↗
PubMed1995

Ligand binding by the IL-2 receptor is modulated by intracellular determinants of the IL-2 receptor beta-chain.

The biologic actions of IL-2 are mediated by the IL-2R, a multisubunit receptor complex displayed on the surface of lymphocytes and select other hematopoietic lineages. The IL-2R exhibits multiple affinities for IL-2 that result from the monomeric (alpha), heterodimeric (alpha beta and beta gamma), and heterotrimeric (alpha beta gamma) assembly of different receptor subunits. In the present studies, we have used a series of IL-2R mutants in a transient mammalian expression system to investigate the potential role of intracellular receptor regions in the ligand-binding functions of the IL-2R. Analyses of chimeric and deletion mutants of the IL-2R beta subunit have revealed that its intracellular domain critically and selectively influences high affinity ligand binding mediated through the extracellular domains of the alpha beta-heterodimeric receptor. In contrast, intermediate affinity binding of IL-2 by beta gamma-heterodimeric receptors exhibits no dependence on the cytoplasmic domain of IL-2 R beta. Further, co-expression of either a full-length or severely truncated form of IL-2 R gamma to generate an alpha beta gamma-heterotrimeric complex also overcomes the functional dependence upon the cytoplasmic tail of IL-2 R beta. Collectively, our findings suggest that the cytoplasmic domain of IL-2R beta produces intrasubunit transmembrane conformational changes in this receptor subunit that promote extracellular IL-2 binding in combination with IL-2R alpha. These findings have important implications for the receptor dynamics involved in both ligand binding and signal transduction as well as for clinical applications pertaining to altering IL-2R function.

Animals↗
PubMed1995

Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef.

Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both cell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity and the downregulation of CD4 represent linked or independent biologic properties of this single protein. In the present studies, mutational analyses were performed to define structure-function relationships within the Nef protein that mediate these effects. To assess the functional consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 downregulation functions of Nef. The results indicate that membrane-targeting sequences at the N terminus of Nef are important for both functions of Nef, while certain other conserved regions are dispensable for both functions. A conserved proline-X-X repeat segment in the central core of the protein, which is reminiscent of an SH3-binding domain, is critical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef appears to involve a two-step process requiring the initial dissociation of p56lck from CD4 to permit engagement of the endocytic apparatus by CD4. Together, these findings demonstrate that the infectivity enhancement and CD4 downregulation activities of human immunodeficiency virus type 1 Nef can be dissociated. Thus, these processes may be independent of one another in the viral replication cycle.

Base Sequence↗
PubMed1994

The cytoplasmic domain of the interleukin-2 receptor beta chain contains both unique and functionally redundant signal transduction elements.

Binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) triggers a series of intracellular events culminating in lymphocyte proliferation and differentiation. A novel transient assay of signal transduction leading to proliferation is now described which allows the rapid functional assessment of wild type and mutant receptors, including the IL-2R and other members of the cytokine receptor superfamily. This assay has been used to define domains and specific residues within the IL-2R beta intracellular region that contribute to growth signal transduction. In these studies, internal deletion of either the conserved "Box 1" or "Box 2" proximal cytokine receptor homology segments significantly impaired receptor function. Similarly, mutation of specific key residues within or between Box 1 and Box 2, or deletion of the C-terminal 94 residues of the IL-2R beta chain, impaired growth signaling. In contrast, either replacement of the transmembrane domain with that of the CD4 molecule or internal deletion of the 119 amino acids immediately downstream of Box 2 had no impact on growth signaling competence. These studies thus further define the functional architecture of the intracellular region of IL-2R beta, and reveal specific receptor domains that are dispensable, unique, or functionally redundant.

Amino Acid Sequence↗
PubMed1990

A transfected human muscarinic receptor fails to substitute for the T cell antigen receptor complex in CD2-initiated signal transduction.

Several T cell surface molecules can activate signal transduction pathways that lead to T cell activation. Like the T cell antigen receptor (TCR), several other molecules, including the sheep erythrocyte receptor CD2, are able to activate the phosphatidylinositol (PI) signal transduction pathway upon stimulation with appropriate agonists. However, CD2-initiated activation of this pathway is dependent on the functional expression of the TCR. Since the T cell does not express other known receptors that activate the PI pathway independent of the TCR, the specificity of the CD2 requirement for a functional TCR is not known. To evaluate the specificity of this requirement, we examined the functional capacity of CD2 to activate the PI pathway in a TCR-deficient cell which had been transfected with a heterologous receptor, the human muscarinic subtype 1 receptor (HM1). HM1 is a member of the cholinergic family of receptors and is known to activate the PI pathway. HM1 can function in the absence of the TCR in a Jurkat-derived T cell host. Here we demonstrate through calcium fluorimetry and PI metabolism assays that HM1 is unable to substitute functionally for the TCR in CD2-initiated signal transduction. These results suggest a specific functional interaction between CD2 and the TCR in CD2-mediated activation of the PI pathway in T cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Function of a heterologous muscarinic receptor in T cell antigen receptor signal transduction mutants.

Previously we have described a system of somatic cell genetics (J.CaM1 and J.CaM2) for analyzing signal transduction via the T cell antigen receptor complex (CD3/Ti). Here we describe a third mutant, J.CaM3, which also expresses high levels of receptors that are functionally impaired. Like J.CaM1, J.CaM3 demonstrates partial signal transduction via CD3/Ti to only certain stimuli. J.CaM1, J.CaM2, and J.CaM3 define three non-Ti complementation groups involved in receptor function. To evaluate the mutations further we have introduced a heterologous receptor, the human muscarinic receptor 1 (HM1), into the parental Jurkat and mutant cell lines. This receptor demonstrates signal transduction competence in all these hosts, indicating that 1) T cells express the necessary apparatus for the coupling of HM1 to second messenger generation and 2) the mutations in the J.CaM family all affect molecules that are specific to CD3/Ti, and not HM1, function. Finally, the HM1 receptor exhibits partial sensitivity to cholera toxin in Jurkat cells, in contrast to the virtually complete sensitivity of CD3/Ti to cholera toxin.

Antibodies, Monoclonal↗
PubMed1989

Ligand-induced association between the T-cell antigen receptor and two glycoproteins.

We have identified two cell surface glycoproteins of 34 and 38 kDa (gp34 and gp38) that associate with the T-cell antigen receptor (TCR). The coimmunoprecipitation of these proteins with the TCR is increased by treatment with monoclonal antibodies (mAbs) directed against the TCR prior to cell solubilization and immunoprecipitation. Treatment of T cells with mAbs directed against other cell surface molecules, CD2 or HLA, does not induce the association of these proteins with the TCR. The coimmunoprecipitation of gp34 and gp38 with the TCR requires solubilization in the presence of an alkylating agent, suggesting that subunit alkylation stabilizes the interaction. J.CaM1 and J.CaM2 are signal-transduction mutant cell lines derived from Jurkat. These cell lines fail to activate the inositol phospholipid second messenger pathway in response to anti-TCR mAbs. Treatment with mAb C305 (anti-TCR) induces the association of gp34 and gp38 with the TCR in J.CaM2 cells but not in J.CaM1. J.CaM1 modulates the TCR normally in response to anti-TCR antibody treatment. This observation suggests that gp34 and gp38 are involved in the signal-transduction pathway of the TCR complex rather than receptor internalization. Furthermore, since these proteins do associate with the TCR of J.CaM2, the induced association with the TCR is not a consequence of signal transduction.

Antibodies, Monoclonal↗
PubMed1989

Molecular characterization of a Leydig cell tumor presenting as congenital adrenal hyperplasia.

We present an unusual patient with a Leydig cell tumor to show that greatly elevated serum concentrations of 17-hydroxyprogesterone (17OHP) may not be diagnostic of congenital adrenal hyperplasia (CAH). A 3.5-yr-old boy had a small testicular mass and plasma 17OHP concentrations of 147-333 nmol/L (4,850-11,000 ng/dL), suggesting CAH with adrenal rests. However, normal plasma cortisol values and the unresponsiveness of the 17OHP concentration to dexamethasone suppression or ACTH stimulation suggested a diagnosis of Leydig cell tumor. A 4-fold elevation in plasma 21-deoxycortisol compared with a 200-fold elevation in 17OHP suggested that the elevated 17OHP derived from the normal pathway of testosterone synthesis in the testis. This was proven by normalization of all hormonal values after tumor resection. Compared to the abundance of mRNA for P450c17, the tumor contained unusually large amounts of mRNA for P450scc, the cholesterol side-chain cleavage enzyme, which is the rate-limiting step in steroid hormone synthesis. Increased P450scc activity, which increased the conversion of cholesterol to pregnenolone, apparently permitted the 17,20-lyase activity of P450c17 to become rate limiting, thus accounting for the increased secretion of 17OHP. Thus, Leydig cell tumors can produce quantities of 17OHP previously reported only in CAH due to 21-hydroxylase deficiency. The molecular characterization of steroidogenic mRNAs in this tumor indicates an unusual ratio in the expression of the genes for the steroidogenic enzymes, probably accounting for the unusual pattern of serum steroids.

17-Ketosteroids↗
PubMed1988

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor.

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.

Adjuvants, Immunologic↗
PubMed1988

Early signal transduction by the antigen receptor without commitment to T cell activation.

The T lymphocyte antigen-receptor complex mediates antigen-specific cell activation, at least in part, through the production of inositolphospholipid-derived second messengers. Little is known about how second messenger events, typically measured within minutes of ligand binding, eventually lead to distal biologic responses such as expression of lymphokine genes. Several monoclonal antibodies directed against the receptor complex were tested for their ability to elicit transmembrane signaling in the parental Jurkat line and in a somatic mutant (J.CaM1) with a deficient receptor function. One antibody elicited substantial early Ca2+ mobilization responses in both cells but was unable to promote expression of the interleukin-2 gene in J.CaM1. In J.CaM1 there was a diminished production of phosphatidylinositol second messengers, and the elevation in intracellular free Ca2+ was transient. Thus, short-term Ca2+ mobilization does not always indicate complete signal transmission and lead to a full cellular response.

Calcium↗
PubMed1988

Function of the antigen receptor in T cell activation.

The studies described here, including ours and those of others, were aimed at understanding the mechanism(s) by which the T cell antigen receptor transmits signals that result in expression of lymphokine genes. They have focused both on the proximal molecules and biochemical events that account for transmembrane signalling, as well as on the more distal nuclear regulatory events. It is now clear that the receptor is coupled to the PI pathway, apparently via conformational changes in Ti, possibly amplification by CD3, and likely a G protein. PI second messengers appear to contribute to subsequent gene expression events, but may not be required for receptor down-regulation. How these biochemical events are coupled to gene expression still remains a mystery. However, studies of the regulation of the IL-2 gene indicate that the proximal receptor-mediated events must occur continuously for 2-4 hours to support transcriptional activation of the IL-2 gene, and that protein synthesis during this time is required. Other genes, such as the proto-oncogene c-fos, become transcriptionally active within 15 minutes of receptor triggering even in the presence of protein synthesis inhibitors. Thus, coupling of the proximal biochemical events to expression of the IL-2 gene may be dependent on the induction of a regulatory protein(s). This protein(s) may act to activate the IL-2 gene via a 275 bp segment that lies upstream of the IL-2 coding region. Studies are now in progress to understand better the molecular requirements for competence in signal transduction and conversion of these signals into gene expression events.

Animals↗
PubMed1988

At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor.

In the T-cell somatic mutant J.CaM1, the T-cell antigen receptor complex is poorly coupled to the inositolphospholipid second messenger system; some antibodies against the invariant CD3 subunit of the receptor retain their agonist function in J.CaM1. Here we show by a combination of complementation assays that the mutation in J.CaM1 affects a molecule other than the antigen-binding Ti subunit, suggesting that Ti is coupled indirectly to the signal transduction apparatus through a pathway involving the CD3 complex. We also describe another mutant, J.CaM2, in which the receptor complex is completely uncoupled from inositolphospholipid hydrolysis. J.CaM2 defines an additional complementation group, suggesting that signal transduction by the antigen receptor depends on at least two molecules distinct from Ti.

Calcium↗