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Interleukin 13 (IL13) belongs to a family of cytokines whose members exhibit structural homology, despite amino acid sequence dissimilarity. For example, while of limited sequence homology, IL13 and IL4 share a signaling receptor, IL13/4 receptor, on a variety of human normal cells. However, a subclass of IL4-independent IL13 receptors is overexpressed on certain transformed cells, including human malignant gliomas. We introduced mutations into human (h) IL13 to determine the site(s) involved in interaction with the shared receptor and/or the glioma-associated receptor. This analysis identified at least three protein regions that are needed for signaling through the shared receptor. These regions were localized to alpha-helices A, C, and D and were mainly separate from the region(s) needed to interact with the glioma-associated receptor. Glutamic acids at positions 13 and 16 in hIL13 alpha-helix A, arginine and serine at positions 66 and 69 in helix C, and arginine at position 109 in helix D were found to be important in inducing biological signaling since their specific mutation resulted in loss and/or gain of function phenomena. We demonstrate that the molecular requirements of hIL13 to interact with its respective receptors are generally distinct and can be controlled by mutagenesis of the cytokine.

A novel subunit of the gamma-aminobutyrate, type A (GABAA) receptor family has been identified in human and rat tissues. The subunit displays 30-40% amino acid identity with known family members and represents a distinct subunit class (termed pi). Transcripts of the pi subunit were detected in several human tissues and were particularly abundant in the uterus. The pi subunit protein can assemble with known GABAA receptor subunits and confer unique ligand binding properties to the recombinant receptors in which it combines. Most notably, the presence of the pi subunit alters the sensitivity of recombinant receptors to the endogenous steroid, pregnanolone. Identification of the pi subunit indicates a new target for pharmacological manipulation of GABAA receptors that are located outside of the central nervous system.

Functional receptors for interleukin (IL)-4 and IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor alpha-chain (IL-4Ralpha) and a 65-75-kDa IL-13 binding subunit that are expressed in a ratio of about 1:3, respectively. The restricted number of IL-4Ralpha limits subunit heterodimerization and in turn receptor-mediated signaling. We report here, the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of the receptor subunits for IL-4 and IL-13. By flow cytofluorometry and receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with TNF-alpha-induced a 2-3-fold increase of the IL-4Ralpha expression. The up-regulation was also confirmed at the transcriptional level by reverse transcription-polymerase chain reaction. Radioligand cross-linking experiments revealed no change in the subunit composition of the TNF-alpha-induced receptor complex. Nevertheless, TNF-alpha stimulation led to increased activation of the IL-4-specific signal transducers and activators of transcription protein (Stat6) by IL-4 and IL-13. Thus, TNF-alpha corrects the subunit imbalance of the endothelial IL-4.IL-13 receptor complex thereby increasing receptor heterodimerization and in turn the signaling capability by IL-4 and IL-13.

The Type I interferon (IFN) receptor has a multisubunit structure. The component of the receptor that has been most thoroughly studied is the alpha subunit. Expression of the alpha subunit in mouse L-929 cells confers antiviral response to human IFN alpha 8, but not to human IFN alpha 2 or IFN beta. This antiviral effect is observed without a significant increase in IFN binding. It has not been determined why mouse cells expressing the human alpha subunit show different response to the antiviral activity of distinct human Type I IFNs. In this report, we demonstrate that the response to human Type I IFNs in mouse cells expressing the alpha subunit is dependent on cross-binding to the mouse receptor. This is supported by the finding that human IFN alpha 8, but not human IFN alpha 2, cross-binds to the mouse receptor even in the absence of expression of the human alpha subunit. We also demonstrate that only mouse cells expressing the human alpha subunit are able to tyrosine-phosphorylate p135tyk2 and JAK-1 and to form the ISGF3 complex in response to human IFN alpha 8. These results demonstrate that the alpha subunit is essential for IFN alpha signaling through the JAK kinases and ISGF3.

We have investigated tyrosine phosphorylation of cellular proteins induced by interleukin (IL) 4 and compared it with the effects of three related cytokines, IL-2, IL-7, and IL-13. We show here that both IL-4 and IL-13 stimulate tyrosine phosphorylation of the 140-kDa IL-4 receptor subunit, which suggests that this receptor protein is used by both cytokines. Receptor phosphorylation induced by IL-13 was both weaker and slower than with IL-4. Stimulation of cells with IL-2 and IL-7 induced identical phosphorylation patterns to each other but not phosphorylation of the 140-kDa IL-4 receptor subunit. The only signal appearing upon stimulation with any of the four cytokines was the weak phosphorylation of an unidentified protein of 160 kDa. SH2 domains of p56 lck and p59 fyn precipitated the same proteins as anti-phosphotyrosine antibodies after IL-4 stimulation, which suggests that a src-type kinase may be involved in signal transduction through the IL-4 receptor.

Interleukin 2 (IL-2) can stimulate the proliferation of various kinds of T-cell lines. The receptor for IL-2 is composed of at least two subunits (alpha and beta), of which beta subunit plays the major role in transducing growth signals into the cells. A nonreceptor-type tyrosine kinase, Lck, is associated with IL-2 receptor beta subunit, and the binding of IL-2 to its receptor induces the activation of Lck. On the other hand, it has been shown that stimulation of T-cells with IL-2 causes rapid activation of Ras protein. In this paper, we describe that both of the two regions in IL-2 receptor beta subunit, the indispensable region for the induction of cell growth (serine-rich region) and the binding region of Lck protein (acidic region), are required for the activation of Ras. These two regions are also required for tyrosine phosphorylation of an 85-kDa cellular protein (p85) and the accumulation of fos and jun mRNAs. This observation suggests also that the activation of a receptor-associated tyrosine kinase in response to IL-2-stimulation is primarily responsible for subsequent activation of the pathway through Ras to Fos and Jun.

The integrins are a large family of heterodimeric cell-surface glycoproteins that play key roles in the adherence of cells to other cells and to extracellular matrix proteins. We have previously reported the identification of a novel integrin beta subunit partial cDNA from leukocytes. We have now determined the complete sequence of this subunit, designated as beta 7, from overlapping clones obtained from a PEER T leukemia cell library and a peripheral T cell library. The beta 7 cDNA contains a single large open reading frame predicted to encode a 798-amino acid protein precursor (signal peptide plus mature protein). The beta 7 protein, like the other beta subunit proteins, is predicted to contain a large extracellular portion, a transmembrane domain, and a cytoplasmic tail. The deduced beta 7 amino acid sequence is 32-46% identical to the six previously sequenced human integrin beta subunits. beta 7 is most similar to the leukocyte integrin common beta subunit (beta 2, CD18). Analysis of variant beta 7 cDNA clones and reverse transcription-polymerase chain reaction products suggest that alternatively spliced beta 7 mRNAs can be generated by the removal of exons that encode most of the cysteine-rich region of the extracellular portion of beta 7. By Northern blot analysis, beta 7 mRNA was detected in T and B cell lines and in macrophage-like cell lines, but not in any of the nonleukocyte cell lines tested. Peripheral T cells and some lymphoma lines express little beta 7 mRNA before stimulation; but after stimulation with phorbol ester, beta 7 mRNA levels increased markedly. Integrin beta 7 is expected to play a role in adhesive interactions of leukocytes.