BOMBESIN RECEPTOR FROM SWISS 3T3-CELLS - AFFINITY-CHROMATOGRAPHY AND RECONSTITUTION INTO PHOSPHOLIPID-VESICLES

被引:8
作者
COFFER, A [1 ]
SINNETTSMITH, J [1 ]
ROZENGURT, E [1 ]
机构
[1] IMPERIAL CANC RES FUND, POB 123, LINCOLNS INN FIELDS, LONDON WC2A 3PX, ENGLAND
来源
FEBS LETTERS | 1990年 / 275卷 / 1-2期
关键词
Biotinylated bombesin; Growth control; Signal transduction;
D O I
10.1016/0014-5793(90)81462-W
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys1]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [13S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed[125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity. © 1990.
引用
收藏
页码:159 / 164
页数:6
相关论文
共 27 条
[1]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[2]  
CARNEY DN, 1987, CANCER RES, V47, P821
[3]   SOLUBILIZATION OF THE BOMBESIN RECEPTOR FROM SWISS 3T3 CELL-MEMBRANES - FUNCTIONAL ASSOCIATION TO A GUANINE-NUCLEOTIDE REGULATORY PROTEIN [J].
COFFER, A ;
FABREGAT, I ;
SINNETTSMITH, J ;
ROZENGURT, E .
FEBS LETTERS, 1990, 263 (01) :80-84
[4]   BOMBESIN-LIKE PEPTIDES CAN FUNCTION AS AUTOCRINE GROWTH-FACTORS IN HUMAN SMALL-CELL LUNG-CANCER [J].
CUTTITTA, F ;
CARNEY, DN ;
MULSHINE, J ;
MOODY, TW ;
FEDORKO, J ;
FISCHLER, A ;
MINNA, JD .
NATURE, 1985, 316 (6031) :823-826
[5]  
DICKEY BF, 1987, J BIOL CHEM, V262, P8738
[6]  
KRIS RM, 1987, J BIOL CHEM, V262, P11215
[7]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+
[8]   CA-2+-MOBILIZING ACTIONS OF PLATELET-DERIVED GROWTH-FACTOR DIFFER FROM THOSE OF BOMBESIN AND VASOPRESSIN IN SWISS 3T3 MOUSE CELLS [J].
LOPEZRIVAS, A ;
MENDOZA, SA ;
NANBERG, E ;
SINNETTSMITH, J ;
ROZENGURT, E .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (16) :5768-5772
[9]   VESICLES OF VARIABLE SIZES PRODUCED BY A RAPID EXTRUSION PROCEDURE [J].
MAYER, LD ;
HOPE, MJ ;
CULLIS, PR .
BIOCHIMICA ET BIOPHYSICA ACTA, 1986, 858 (01) :161-168
[10]   EARLY EVENTS ELICITED BY BOMBESIN AND STRUCTURALLY RELATED PEPTIDES IN QUIESCENT SWISS 3T3 CELLS .2. CHANGES IN NA+ AND CA-2+-FLUXES, NA+/K+ PUMP ACTIVITY, AND INTRACELLULAR PH [J].
MENDOZA, SA ;
SCHNEIDER, JA ;
LOPEZRIVAS, A ;
SINNETTSMITH, JW ;
ROZENGURT, E .
JOURNAL OF CELL BIOLOGY, 1986, 102 (06) :2223-2233