Analysis of the N-terminal binding domain of G(0)alpha

被引:14
作者
Busconi, L [1 ]
Denker, BM [1 ]
机构
[1] HARVARD UNIV,SCH MED,BOSTON,MA 02115
关键词
D O I
10.1042/bj3280023
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
Signalling from membrane receptors through heterotrimeric G-proteins (G alpha and G beta gamma) to intracellular effecters is a highly regulated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defined mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of G(o)a (D[11-14]); Busconi, Boutin and Denker (1997) Biochem, J. 323, 239-244}. We characterized N-terminal mutants of G(o) alpha with known G beta gamma-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of G alpha to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal alpha-helix of G(o) alpha with a proline point mutation at Arg-9 (R9P) does not affect interactions with G beta gamma on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced G beta gamma binding) or deletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type G(o) alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of G(o) alpha and mutants translated in vitro to liposomes indicates that Go alpha preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like G(o) alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of G alpha subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for regulating signal transduction.
引用
收藏
页码:23 / 31
页数:9
相关论文
共 51 条
[2]  
BOURNE HR, 1991, NATURE, V349, P117, DOI 10.1038/349117a0
[3]   THE GTPASE SUPERFAMILY - A CONSERVED SWITCH FOR DIVERSE CELL FUNCTIONS [J].
BOURNE, HR ;
SANDERS, DA ;
MCCORMICK, F .
NATURE, 1990, 348 (6297) :125-132
[4]   LOCALIZATION AND POLAR DISTRIBUTION OF SEVERAL G-PROTEIN SUBUNITS ALONG NEPHRON SEGMENTS [J].
BRUNSKILL, N ;
BASTANI, B ;
HAYES, C ;
MORRISSEY, J ;
KLAHR, S .
KIDNEY INTERNATIONAL, 1991, 40 (06) :997-1006
[5]   N-terminal binding domain of G alpha subunits: Involvement of amino acids 11-14 of G alpha(o) in membrane attachment [J].
Busconi, L ;
Boutin, PM ;
Denker, BM .
BIOCHEMICAL JOURNAL, 1997, 323 :239-244
[6]  
BUSCONI L, 1994, J BIOL CHEM, V269, P25016
[7]   MEMBRANE-BINDING OF MYRISTYLATED PEPTIDES CORRESPONDING TO THE NH2 TERMINUS OF SRC [J].
BUSER, CA ;
SIGAL, CT ;
RESH, MD ;
MCLAUGHLIN, S .
BIOCHEMISTRY, 1994, 33 (44) :13093-13101
[8]   CYTOSKELETAL CONSTRAINT OF THE BETA-ADRENERGIC-RECEPTOR IN FROG ERYTHROCYTE-MEMBRANES [J].
CHERKSEY, BD ;
ZADUNAISKY, JA ;
MURPHY, RB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1980, 77 (11) :6401-6405
[9]   STRUCTURES OF ACTIVE CONFORMATIONS OF G(I-ALPHA-1) AND THE MECHANISM OF GTP HYDROLYSIS [J].
COLEMAN, DE ;
BERGHUIS, AM ;
LEE, E ;
LINDER, ME ;
GILMAN, AG ;
SPRANG, SR .
SCIENCE, 1994, 265 (5177) :1405-1412
[10]  
DEALMEIDA JB, 1994, J CELL SCI, V107, P507