Tài liệu Báo cáo Y học: The Fc receptor c-chain is necessary and sufficient to initiate signalling

The Fc receptor c-chain is necessary and sufficient to initiate

signalling through glycoprotein VI in transfected cells by the snake

C-type lectin, convulxin

Oscar Berlanga1,2, David Tulasne1

, Teresa Bori3

, Daniel C. Snell1

, Yoshiki Miura4

, Stephanie Jung4

,

Masaaki Moroi4

, Jonathan Frampton2 and Steve P. Watson1

1

Department of Pharmacology, University of Oxford, UK; 2

Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,

Oxford, UK; 3

Division of Medical Sciences, The Medical School, University of Birmingham, UK; 4

Department of Protein

Biochemistry, Institute of Life Sciences, Kurume University, Japan

There is extensive evidence that FcR c-chain couples to the

collagen receptor glycoprotein VI (GPVI)and becomes

phosphorylated on tyrosines upon receptor cross-linking.

However, it is not established whether this receptor complex

is sufficient to initiate the signalling cascade. We transfected

GPVI and the FcR c-chain into the human erythroleukae￾mia cell line K562, which lacks detectable expression of

GPVI and the FcR c-chain. The results show that GPVI is

unable to signal when expressed alone, despite its surface

expression, upon stimulation with the snake C-type lectin,

convulxin. Coexpression of the FcR c-chain confers

signalling properties on the receptor. Furthermore,

cotransfection of the FcR c-chain and two mutant versions

of GPVI shows that the transmembrane arginine and cyto￾plasmic tail of GPVI are necessary for association with the

FcR c-chain. These results demonstrate that reconstitution

of the GPVI–FcR c-chain complex in cells expressing the

necessary signalling network is sufficient to initiate signalling

events in response to convulxin and collagen-related peptide.

Keywords: collagen; collagen-related peptide (CRP);

convulxin; FcR c-chain; glycoprotein VI (GPVI).

Glycoprotein VI (GPVI)is the major signalling collagen

receptor present in platelets and megakaryocytes. It

belongs to the superfamily of immunoblobulin receptors

and is closely related to Fca receptor (FcaR)and natural

killer receptor [1]. The cloned cDNA of GPVI indicates

an ORF encoding a 20 amino-acid signal sequence and a

mature protein of 319 amino acids. Its extracellular

region has two immunoglobulin-like domains and a

mucin-like Ser/Thr region, followed by a transmembrane

and short cytoplasmic tail. GPVI forms a complex with

the Fc receptor c-chain (FcR c-chain), which is respon￾sible for signalling through GPVI [2–4]. Previous reports

on two receptors sharing homology with GPVI, namely

FcaR and paired immunoglobulin-like receptor A (PIR￾A), revealed that they are expressed on the surface of the

membrane independently of the FcR c-chain in cell lines

[5,6], but coexpression with FcR c-chain increases the

level of expression of the receptor at the surface [5,7].

The interaction between FcaR or PIR-A and the FcR

c-chain occurs in the transmembrane region as the result

of oppositely charged amino-acid residues [6,8]. The

immune receptor tyrosine-based activation motif domain

within the cytoplasmic tail of the FcR c-chain is

responsible for signalling after engagement of the recep￾tor complex [9].

The interaction between platelets and collagen involves

adhesion and activation leading to increased strength of

adhesion, secretion and ultimately aggregation [10–12]. It is

accepted that the integrin a2b1 is the major receptor

supporting strong platelet adhesion to collagen, whereas

GPVI mediates activation [3,13]. The multimeric nature of

collagen means that the development of specific ligands to

either receptor is essential for understanding their relative

contribution to the overall mechanism of platelet–collagen

interaction. Among these, collagen-related peptide (CRP)is

thought to signal specifically through GPVI, as demonstra￾ted by the lack of response to the peptide in GPVI-deficient

platelets [13]. Convulxin, a C-type lectin from the venom of

the tropical rattlesnake Crotalus durissus terrificus, also

specifically recognizes GPVI.

The present results show surface expression of GPVI

independently of the FcR c-chain in COS-7 and K562 cells,

and that the transmembrane arginine and cytoplasmic

domain of GPVI are necessary for association with the FcR

c-chain. Moreover, the FcR c-chain is necessary and

sufficient to initiate the signalling events after GPVI

engagement, as demonstrated for the first time in a

reconstituted system in which both GPVI and FcR c-chain

have been stably expressed.

Correspondence to O. Berlanga, Weatherall Institute of Molecular

Medicine, University of Oxford, John Radcliffe Hospital,

Headington, Oxford OX3 9DS, UK.

Fax:+ 44 1865 222737, Tel.: + 44 1865 222437,

E-mail: [email protected]

Abbreviations: GPVI, glycoprotein VI; FcRc, Fc receptor c-chain;

CRP, collagen-related peptide; FITC, fluorescein isothiocyanate;

GFP, green fluorescent protein; GST, glutathione S-transferase;

PIR-A, paired immunoglobulin-like receptor A; PMA,

phorbol myristate acetate.

(Received 14 November 2001, revised 20 March 2002,

accepted 30 April 2002)

Eur. J. Biochem. 269, 2951–2960 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02969.x