α1B-adrenoceptor interacts with multiple sites of transglutaminase II:: Characteristics of the interaction in binding and activation

被引:32
作者
Feng, JF [1 ]
Gray, CD [1 ]
Im, MJ [1 ]
机构
[1] Cleveland Clin Fdn, Lerner Res Inst, Dept Mol Cardiol, Cleveland, OH 44195 USA
关键词
D O I
10.1021/bi9823176
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
We previously reported that a novel GTP binding protein (G alpha(h)) is tissue type transglutaminase (TGII) and transmits the alpha(1B)-adrenoceptor (AR) signal to phospholipase C (PLC) through its GTPase function. We have also shown that PLC-delta 1 is the effector in TGII-mediated signaling. In this study, interaction sires on TGII for the alpha(1B)-AR were identified using a peptide approach and site-directed mutagenesis, including in vivo reconstitution of TGIIs with the alpha(1B)-AR and PLC-delta 1. To identify the interaction sites, 11 synthetic peptides covering similar to 132 amino acid residues of the C-terminal domain of TGII were tested. The studies with the peptides revealed that three peptides, L-547-I-561, R-564-D-581, and Q(633)-E-646, disrupted formation of an alpha(1)-agonist-alpha(1B)-AR-TGII complex and blocked alpha(1B)-AR-mcdlated TGase inhibition in a dose-dependent manner, indicating that these peptide regions are involved in recognition and activation of TGII by the alpha(1B)-AR. These three regions were further evaluated with full-length TGIIs by constructing and coexpressing each site-directed mutant with the alpha(1B)-AR and PLC-delta 1 in COS-1 cells. Supporting the findings with these peptides, these TGII mutants lost 56-82% the receptor binding ability and reduced by 29-68% the level of alpha(1B)-AR-mediated IP3 production via PLC-delta 1 as compared to those with wild-type TGII. The results also revealed that the regions of R-564-D-581 and Q(633)-E-646 were the high-affinity binding sites of TCII for the receptor and critical for the activation of TGII by the receptor. Taken together, the studies demonstrate that multiple regions of TGII interact with the alpha(1B)-AR and that the alpha(1B)-AR stimulates PLC-delta 1 via TGII.
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页码:2224 / 2232
页数:9
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