α1B-Adrenoceptor Signaling and Cell Motility: GTPase FUNCTION OF Gh/TRANSGLUTAMINASE 2 INHIBITS CELL MIGRATION THROUGH INTERACTION WITH CYTOPLASMIC TAIL OF INTEGRIN α SUBUNITS

Sung Koo Kang,Kye Sook Yi,Nyoun Soo Kwon,Kwang-Hyun Park,Uh-Hyun Kim,Kwang Jin Baek,Mie-Jae Im

doi:10.1074/jbc.m402084200

Sung Koo Kang, Kye Sook Yi + Show 5 more

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https://doi.org/10.1074/jbc.m402084200

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Abstract

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

Highlights

A multifunctional enzyme, Gh, is a GTP-binding protein that couples to the ␣1B-adrenoreceptor and stimulates phospholipase C-␦1 and displays transglutaminase 2 (TG2) activity
Interaction of Integrin ␣5 Cytoplasmic Tail with GTP␥Sbound Gh/TG2 Modulates Cell Migratory Activity—To identify the signaling molecule(s) targeted by Gh/TG2 in inhibition of cell migration, we examined whether Gh/TG2 interacts with a prototype Fn receptor, integrin ␣5␤1
Because the above results suggested that intracellular Gh/TG2 is involved in modulation of cell migration and integrin ligation is critical in this cellular response, coimmunoprecipitation studies were performed using integrin ␣5 subunit antibody against extracellular domain under various conditions; cell extract prepared from HEK 293 cell-overexpressed wtTG was incubated in the presence and absence of guanosine 5؅-3O-(thio)triphosphate (GTP␥S) or a soluble ligand of Fn, a 120-kDa fragment that contains an RGD motif but not the Gh/TG2 binding site or a known blocking ligand (RGD peptide) or non-blocking ligand (RGE peptide) of integrins

Summary

Introduction

A multifunctional enzyme, Gh, is a GTP-binding protein that couples to the ␣1B-adrenoreceptor and stimulates phospholipase C-␦1 and displays transglutaminase 2 (TG2) activity. The results indicate that modulation of the cell migratory activity is mediated by the binding of Gh to the GFFKR motif present in the cytoplasmic tail of various integrin ␣ subunits.

Results

Conclusion