Functional contributions of the FcepsilonRIalpha and FepsilonRIgamma subunit domains in FcepsilonRI-mediated signaling in mast cells.

The functional contributions of the alpha and gamma subunit domains of the high affinity receptor for IgE (Fcepsilon-RI) were determined following chimeric receptor aggregation. Chimeric receptors of the extracellular (EC) and cytoplasmic tail (CT) domains of FcepsilonRI and the IL-2R p55 subunit (I) were constructed and stably expressed in RBL-2H3 cells. Signaling (inositol phosphate production, tyrosine phosphorylation, Ca2+ mobilization, and secretion of histamine and arachidonic acid metabolites) via alpha/gamma/gamma or I/gamma/gamma was similar to the native rat receptor, and both were shown to associate with endogenous FcepsilonRIbeta and FcepsilonRIgamma subunits. Therefore, the contributions of the EC domains could not be evaluated. The chimeras alpha/I/gamma and I/I/gamma were found to be single polypeptide chains, as they did not associate with beta and gamma. Signaling via alpha/I/gamma resulted in the appearance of biochemical events common to the native receptor. Cross-linking I/I/gamma elicited histamine release, [14C]arachidonic acid metabolites, tyrosine phosphorylation, Ca2+ mobilization, and only inositol trisphosphate production, which were not of a similar magnitude to the native FcepsilonRI. No biochemical events were elicited by cross-linking alpha/I/I or I/I/I. These results demonstrate that both the FcepsilonRIalpha EC domain and the FcepsilonRIgamma CT domain are essential for the FcepsilonRI signaling process, and that while FcepsilonRIIgamma CT plays a critical role in FepsilonRI signaling, the EC domain of FcepsilonRIalpha has a major contribution in signaling, as well as a role in modulating the magnitude of the biochemical events.

Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.

A novel strategy for secreting proteins: use of phosphatidylinositol-glycan-specific phospholipase D to release chimeric phosphatidylinositol-glycan anchored proteins.

Phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) specifically hydrolyzes the inositol-phosphate linkage in phosphatidylinositol-glycan (PI-G) anchored proteins. We recently deduced the primary structure of this enzyme and demonstrated specific enzymatic activity in transfected cells. Co-transfection of PI-G PLD with a natural PI-G anchored protein resulted in the secretion of the PI-G anchored protein via a PI-G PLD specific mechanism. We have taken advantage of these observations to develop an alternative system that may be useful for expressing and secreting proteins not amenable to secretion by conventional methods. Chimeric PI-G anchored proteins were constructed by transferring the COOH-terminal signal peptide for PI-G anchor attachment from placental alkaline phosphatase or from the low affinity IgG receptor, FcGRIIIB, to proteins that are not normally PI-G anchored. This process facilitates the cell surface expression of several proteins including the high affinity IgE receptor alpha subunit, FcERI alpha, which otherwise requires at least one other subunit for surface expression. Co-expression of these chimeric PI-G anchored proteins with PI-G PLD resulted in their secretion via a PI-G PLD specific mechanism.

Association of the human Fc epsilon RI gamma subunit with novel cell surface polypeptides.

It has recently been demonstrated that the gamma-subunits of the high affinity Fc epsilon RI are required for the cell surface expression of not only the Fc epsilon RI alpha-subunit, but also for the low affinity Fc gamma RIIIA alpha (CD16). In addition, formation of heterodimeric complexes of the gamma-subunit with the zeta- and eta-chains of the TCR have also been reported. The exact role of the gamma-subunit in the function of these receptors is not known. To gain additional insight into the association of the gamma-subunit with these and other cell surface polypeptides, we have generated a mAb, 4D8, directed against the human Fc epsilon RI gamma-subunit. Using this antibody we have been able to demonstrate that Fc epsilon RI alpha and Fc gamma RIIIA alpha are associated with Fc epsilon RI gamma at the cell surface. Furthermore, we have identified the expression of Fc epsilon RI gamma in HL60 and U937 cells, which are negative for the TCR, Fc epsilon RI, and Fc gamma RIII. Analysis of these cells reveals the presence of novel Fc epsilon RI gamma-associated polypeptides. These results suggest that Fc epsilon RI gamma plays a common functional role in a number of different receptor complexes. The availability of the anti-gamma antibody 4D8 will help to define this role, and allow the characterization of cell surface polypeptides that are associated with the Fc epsilon RI gamma-subunit.

Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D.

A phosphatidylinositol-glycan-specific phospholipase D (PI-G PLD) that specifically hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol-glycans (PI-Gs) has recently been purified from human and bovine sera. The primary structure of bovine PI-G PLD has now been determined and the functional activity of the enzyme has been studied. Expression of PI-G PLD complementary DNA in COS cells produced a protein that specifically hydrolyzed the inositol phosphate linkage of the PI-G anchor. Cotransfection of PI-G PLD with a PI-G-anchored protein resulted in the secretion of the PI-G-anchored protein. The results suggest that the expression of PI-G PLD may influence the expression and location of PI-G-anchored proteins.

Administration of recombinant enkephalinase (neutral endopeptidase) prevents capsaicin-induced miosis in the rabbit eye in vivo.

Substance P induced a dose-dependent contraction of iris sphincter muscles when applied in the presence of atropine to the isolated rabbit iris in vitro as evidenced by a decreased pupil diameter. Pretreatment of the iris with 20 micrograms of recombinant enkephalinase (neutral endopeptidase; EC 3.4.24.11) totally abolished the contractile response to substance P. Injection of 10 micrograms of capsaicin into the anterior chamber of atropine-treated rabbit eyes in vivo induced an immediate and intense miosis. Injection of 100 micrograms of recombinant enkephalinase, 1 or 5 min before capsaicin injection, significantly inhibited this miosis. This effect of enkephalinase was totally abolished by preincubating the enzyme with thiorphan, a high-affinity enkephalinase inhibitor. These results show that enkephalinase, which is known to hydrolyze substance P in vitro with high efficiency, also hydrolyzes endogenously released substance P in vivo. Furthermore, our results suggest that enkephalinase application may represent a novel therapeutic approach to treat substance P-mediated pathologies.

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines.

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.

Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases.

Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney. A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library. This allowed the identification of several overlapping clones encoding the full sequence of the enzyme. The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide. A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme. The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site. In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases. Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E. coli (18%) and with human leukotriene A4 hydrolase (21%).

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells.

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.