Distinct Functions of the FcR1 and Subunits in the Control of FcR1-mediated Tyrosine Kinase Activation and Signaling Responses in RBL-2H3 Mast Cells

doi:10.1074/jbc.270.8.4013

Distinct Functions of the FcR1 and Subunits in the Control of FcR1-mediated Tyrosine Kinase Activation and Signaling Responses in RBL-2H3 Mast Cells (*)

From the Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
Basel Institute for Immunology, CH-4058 Basel, Switzerland

^¶ To whom correspondence should be addressed:
Cell Pathology Laboratory, Surge Bldg., University of New Mexico School of Medicine, Albuquerque, NM 87131.
Tel.: 505-277-4364; Fax: 505-277-2999.

Abstract

In RBL-2H3 rat tumor mast cells, cross-linking the high affinity IgE receptor, FcR1, activates the protein-tyrosine kinases Lyn and Syk and initiates a series of responses including protein-tyrosine phosphorylation, inositol 1,4,5-trisphosphate synthesis, Ca mobilization, secretion, membrane ruffling, and actin plaque assembly. The development of chimeric receptors containing cytoplasmic domains of individual subunits of the heterotrimeric (αβ₂) FcR1 has simplified analyses of early signaling events in RBL-2H3 cells. Here, RBL-2H3 cells were transfected with cDNAs encoding the extracellular and transmembrane domains of the interleukin-2 receptor α subunit (the Tac antigen) joined to the C-terminal cytoplasmic domains of the FcR1 and β subunits (TT and TTβ). Both sequences contain tyrosine activation motifs implicated in antigen receptor signal transduction. TT and TTβ are expressed independently of the native FcR1, as demonstrated by the ability of Tac cross-linking agents to trigger the clustering and internalization through coated pits of both chimeric receptors without co-clustering the FcR1. A full range of signaling activities is induced by TT cross-linking; the TT-induced responses are slower and, except for Lyn activation, smaller than the FcR1-induced responses. In striking contrast, TTβ cross-linking elicits no tyrosine phosphorylation or signaling responses, it impairs basal activities measured in secretion and anti-PY (anti-phosphotyrosine antibody) immune complex kinase assays, and it antagonizes FcR1-induced Lyn and Syk activation, protein-tyrosine phosphorylation, and signaling responses. We hypothesize that the isolated β subunit binds a specific kinase or coupling protein(s) required for signaling activity, sequestering it from the signal-transducing subunit. Binding the same kinase or coupling protein to the β subunit of the intact FcR1 may serve instead to present it to the adjacent subunit, resulting in enhanced kinase activation and signaling responses.

Footnotes

↵^§ Supported by a postdoctoral fellowship from the University of New Mexico Cancer Research and Treatment Center.
↵* This work was supported in part by Grant IM-68438 from the American Cancer Society and by National Institutes of Health Grant GM49814. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

FcR1

the high affinity receptor for IgE

anti-PY

anti-phosphotyrosine antibody

DNP

dinitrophenol

BSA

bovine serum albumin

FBS

fetal bovine serum

FITC

fluorescein isothiocyanate

1,4,5-IP₃

inositol 1,4,5-trisphosphate

LRSC

lissamine rhodamine sulfonyl chloride

PAGE

polyacrylamide gel electrophoresis

RBL-2H3

the 2H3 secreting subline of rat basophilic leukemia (RBL) cells

TAM

tyrosine activation motif

TCR

T cell receptor

MEM

minimal essential medium

PBS

phosphate-buffered saline.
↵²R. J. Lee and J. M. Oliver, submitted for publication.
- Received September 8, 1994.
- Revision received November 14, 1994.