Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function.
T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in beta1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the beta1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human beta1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the beta1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the beta1 integrin with the activating beta1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the beta1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of beta1 integrin structure and function in human T cells.
Romzek
N C
NC
Department of Laboratory Medicine and Pathology, Center for Immunology and Cancer Center, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Harris
E S
ES
Dell
C L
CL
Skronek
J
J
Hasse
E
E
Reynolds
P J
PJ
Hunt
S W
SW
Shimizu
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002448:Cell Adhesion; D017931:DNA Primers; D005353:Fibronectins / Q000502:physiology; D006801:Humans; D019012:Integrin beta1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013601:T-Lymphocytes / Q000502:physiology*; D014162:Transfection; D014443:Tyrosine
D017931:DNA Primers; D005353:Fibronectins; D019012:Integrin beta1; D011994:Recombinant Proteins; D014443:Tyrosine