Immunodetection of the Ligand-Activated Receptor for Epidermal Growth Factor

被引:31
作者
Campos-Gonzalez, Roberto [1 ]
Glenney, John R., Jr.
机构
[1] Univ Kentucky, Dept Biochem, Room 206,Combs Res Bldg,800 Rose St, Lexington, KY 40536 USA
关键词
Anti-phosphotyrosine; monoclonal antibodies; EGF receptor internalization;
D O I
10.3109/08977199109043916
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Many receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immunocytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates.
引用
收藏
页码:305 / 316
页数:12
相关论文
共 38 条
[1]   DOWN-REGULATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR IN KB CELLS IS DUE TO RECEPTOR INTERNALIZATION AND SUBSEQUENT DEGRADATION IN LYSOSOMES [J].
BEGUINOT, L ;
LYALL, RM ;
WILLINGHAM, MC ;
PASTAN, I .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (08) :2384-2388
[2]   PHORBOL ESTERS INDUCE TRANSIENT INTERNALIZATION WITHOUT DEGRADATION OF UNOCCUPIED EPIDERMAL GROWTH-FACTOR RECEPTORS [J].
BEGUINOT, L ;
HANOVER, JA ;
ITO, S ;
RICHERT, ND ;
WILLINGHAM, MC ;
PASTAN, I .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (09) :2774-2778
[3]   SPATIAL PATTERN OF MYOSIN PHOSPHORYLATION IN CONTRACTING SMOOTH-MUSCLE CELLS - EVIDENCE FOR CONTRACTILE ZONES [J].
BENNETT, JP ;
CROSS, RA ;
KENDRICKJONES, J ;
WEEDS, AG .
JOURNAL OF CELL BIOLOGY, 1988, 107 (06) :2623-2629
[4]  
BERTICS PJ, 1985, J BIOL CHEM, V260, P4642
[5]   EPIDERMAL GROWTH-FACTOR RECEPTOR IS DOWN-REGULATED BY A 10,400-MW PROTEIN ENCODED BY THE E3 REGION OF ADENOVIRUS [J].
CARLIN, CR ;
TOLLEFSON, AE ;
BRADY, HA ;
HOFFMAN, BL ;
WOLD, WSM .
CELL, 1989, 57 (01) :135-144
[6]   RECEPTORS FOR EPIDERMAL GROWTH-FACTOR AND OTHER POLYPEPTIDE MITOGENS [J].
CARPENTER, G .
ANNUAL REVIEW OF BIOCHEMISTRY, 1987, 56 :881-914
[7]   DIRECT VISUALIZATION OF THE PHOSPHORYLATED EPIDERMAL GROWTH-FACTOR RECEPTOR DURING ITS INTERNALIZATION IN A-431 CELLS [J].
CARPENTIER, JL ;
WHITE, MF ;
ORCI, L ;
KAHN, RC .
JOURNAL OF CELL BIOLOGY, 1987, 105 (06) :2751-2762
[8]   SUBCELLULAR-DISTRIBUTION OF THE EXTERNAL AND INTERNAL DOMAINS OF THE EGF RECEPTOR IN A-431 CELLS [J].
CARPENTIER, JL ;
REES, AR ;
GREGORIOU, M ;
KRIS, R ;
SCHLESSINGER, J ;
ORCI, L .
EXPERIMENTAL CELL RESEARCH, 1986, 166 (02) :312-326
[9]   REQUIREMENT FOR INTRINSIC PROTEIN TYROSINE KINASE IN THE IMMEDIATE AND LATE ACTIONS OF THE EGF RECEPTOR [J].
CHEN, WS ;
LAZAR, CS ;
POENIE, M ;
TSIEN, RY ;
GILL, GN ;
ROSENFELD, MG .
NATURE, 1987, 328 (6133) :820-823
[10]   FUNCTIONAL INDEPENDENCE OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR FROM A DOMAIN REQUIRED FOR LIGAND-INDUCED INTERNALIZATION AND CALCIUM REGULATION [J].
CHEN, WS ;
LAZAR, CS ;
LUND, KA ;
WELSH, JB ;
CHANG, CP ;
WALTON, GM ;
DER, CJ ;
WILEY, HS ;
GILL, GN ;
ROSENFELD, MG .
CELL, 1989, 59 (01) :33-43