Vol.

171, No.

September

3, 1990 28,

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AND

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RESEARCH

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THE MONOCLONAL ANTIBODY AG-1, A POTENT STIMULATOR OF HUMAN PLATELETS, INTERACTS WITH A LOW MOLECULAR WEIGHT GTP-BINDING PROTEIN Michael

H. Kroll*§,

Rebecca

SHematology Baylor aDepartment

Section, College of

E. Claures,

and

Jonathan

L. Milleroo,

Houston VA Medical Center of Medicine, Houston, TX

Pathology, SUNY Health Syracuse, NY

Science

and Center,

Received August 17, 1990 SUMMARY: AG-1 is a monoclonal antibody that binds to human platelets Previous work has established and causes aggregation and secretion. that these responses result from phospholipase C-mediated hydrolysis To determine the of phosphatidylinositol 4,5-bisphosphate (PIP,). mechanism by which this ligand induces signals for platelet activation, we performed a series of experiments examining the AG-1 immunoprecipitates from platelet binding site for AG-1. radioiodinated human platelet plasma membranes a protein of Mr 21,000. AG-1 immunoprecipitated proteins separated by SDS-PAGE, transferred to and incubated with [a32PJGTP demonstrate binding of nitrocellulose, A loo-fold molar the radiolabeled GTP to the Mr 21,000 protein. excess of unlabeled GTP inhibits completely this binding of [a3'P]GTP. These results indicate that AG-1 interacts with a low Mr GTP-binding protein on the surface of platelets and suggests that either the protein recognized by AG-1 or a coprecipitating molecule of similar Mr is a low Mr GTP-binding protein that may function in platelet extracellular signal transduction. 0 1990 Academic Press, Inc.

AG-1 is a murine monoclonal IgG antibody that causes platelet activation (1). It was generated by immunizing mice with washed platelets from patients with platelet-type von Willebrand disease, a rare bleeding disorder characterized by increased affinity between the platelet glycoprotein (Gp) Ib complex and normal von Willebrand factor Its binding to normal resting platelets stimulates aggregation (2) * and secretion (1). These functional responses result from AG-1 induced activation of platelet phospholipase C (PLC) causing phosphatidylinositol 4,5-bisphosphate (PIP,) hydrolysis, protein kinase C activation, and intracellular calcium release (3). *To whom correspondence should be addressed at: Hematology Baylor College of Medicine, MS 902, 6565 Fannin, Houston, 77030.

0006-291X/90 $1.50 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

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Human platelets contain different GTP binding (G) proteins. The best characterized platelet G proteins are dissociable heterotrimeric molecules that couple receptor occupancy to effector enzymes such as PLC or adeny:Lyl cyclase (4). There are, in addition, smaller membrane-associated platelet G proteins whose function are unknown. These low Mr G proteins from human platelets have a size range of Mr 21,000 to 29,500 (5-11). Immunoblots of a Mr 21,000 platelet G protein bind anti-ras p21 antibodies (9,ll). A Mr 22,000 platelet G protein has amino acid sequence homology to bovine brain Sms ~21, whose function is unknown, and may be a substrate for ADP ribosylation by botulinum toxin (7,9,10). This latter phenomenon is associated with the potentiation of the secretion response of stimulated platelets (7). No further structural or functional information is currently available concerning platelet low Mr G proteins. The present report describes experiments examining the hypothesis that AG-1 induced platelet activation results from its interacting with a low Mr G protein. Data from these studies demonstrate that AG1 immunoprecipitates a Mr 21,000 platelet membrane protein that binds GTP. This suggests that platelet membranes contain a low Mr G protein that may function in extracellular signal transduction initiated by the ligand AG-1. METHODS AND MATERIALS [o~~P]GTP and "'I(Na salt) were from New England Nuclear, Boston, MA. Unlabeled GTP was from Boehringer Mannheim, Indianapolis, IN. Monoclonal antibody lOE5 was from Dr. B. Coller, SUNY, Stony Brook, NY. Lactoperoxidase was from Calbiochem, La Jolla, CA. Peroxidase conjugated anti-mouse IgG, Amido Black lOB, and NP-40 were from Sigma, St. Louis, MO. Molecular weight standards were from BioRad, Richmond, CA. Mouse monoclonal antibody AG-1 was produced and purified as previously described (1). Radiolabelincc

and Fractionation of Platelets. Whole blood was with 15% (v/v) ACD and centrifuged at 180 x g for 12 min. Platelet-rich plasma was acidified to pH 6.5, ll.rM PGE, added, and the platelets centrifuged at 800 x g for 10 min. The platelet pellet was washed with 1OmM Tris HCl, 1mM EDTA, 145mM NaCl, pH 7.4, and radiolabeled with Na'"1 by the lactoperoxidase method as previously described (1). Radiolabeled, or unlabeled washed platelets, were detergent-lysed and ultracentrifuged at 100,000 x g for 1 hr, and the resulting plasma membrane-enriched supernatant stored at -7O'C, as previously described (1). anticoagulated

Protein Immunonrecinitation. Sewaration. and Transfer to B. 7.51.14 AG-1 was added to the lysate from lo8 platelets and the protein immunoprecipitated by goat anti-mouse IgG conjugated Following an 18h incubation at 4'C, the beads were to agarose beads. 0.1% NP-40, pH 7.2, washed five times with 1OmM Tris HCl, 140mM NaCl, and the AG-1 bound protein eluted with 5% SDS, 5% glycerol, 1OmM Tris HCl, pH 6.8. 1253

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AG-1 immunoprecipitated SDS-PAGE on 5-15% gradient

protein from lo7 platelets was separated gels as described by Laemmli (12). Proteins were transferred from these gels to nitrocellulose in a Hoefer TE50 apparatus using 25mM Tris HCl, 192 mM glycine, 20% by Towbin et al. (13). methanol (v/v), pH 8.3, as described

by

ra3'P1 GTP Bindins Assav. AG-1 immunoprecipitated platelet lysates transferred to nitrocellulose were incubated in binding buffer (50mM Tris HCl, pH 7.5 containing 0.3% Tween 20, 5mM, MgCl,, and 1mM EGTA) to which was added 50 pci [a32P] GTP (3000 Ci/mmole) for 1.5 hrs at 22v. The nitrocellulose was washed three times in binding buffer, dried, and autoradiographed as described by Bhullar and Haslam (5). For binding inhibition experiments, the nitrocellulose was incubated with 50 PCi [a3'P] GTP in the presence of a loo-fold molar excess of non-radioactive GTP. Proteins transferred to nitrocellulose were Immunoblot Assav. incubated in blocking buffer (1Omm Tris HCl, 140mM NaCl, pH 7.2 plus 5% fetal calf serum and 0.05% Tween 20) for 1 hr at 22'C, washed three times in blocking buffer without fetal calf serum, and then incubated in blocking buffer with antibody (AG-1 = al.cg/ml; lOE5 = 2 pg/ml) for The immunoblots were washed three times and developed l-2 hr at 22'C. with a peroxidase-conjugated goat anti-mouse IgG reacted with 0.06% H,O, in buffer containing 0.05% 4-chloro-l-naphthol.

RESULTS

AND DISCUSSION

Initial experiments were directed at identifying the protein immunoprecipitated from the platelet lysate by AG-1. The first lane of figure 1 shows nitrocellulose stained by Amido Black 10B following the electrophoretic transfer of AG-1 immunoprecipitated proteins This lane demonstrates separated by SDS-PAGE on 5-15% gradient gels. The results a broad band at Mr 21,000 and a second band at Mr 36,000. of these experiments are consistent with previous observations (1). platelet surface proteins Autoradiography of 12'1-labeled immunoprecipitated by AG-1 reveals only the Mr 21,000 band (Figure llane 2). The absence of the Mr 36,000 band from the '251-labeled immunoprecipitate suggests that the Mr 21,000 protein may be on the outer surface of the platelet plasma membrane, while the Mr 36,000 protein may be located within the inner aspect of the platelet plasma membrane and therefore not accessible to radioiodinization. The third lane of figure 1 shows that both Mr 21,000 and Mr 36,000 bands bind the AG-1 antibody. Previous data have established that the Mr 36,000 band stains nonspecifically on immunoblotting (1). To confirm this previous observation, blots were incubated with the monoclonal antibody lOE5, which binds to platelet integrin molecules, including glycoprotein IIb-IIIa (14). lOE5 was observed to bind strongly to the Mr 36,000 protein but only weakly to the Mr 21,000 band (not shown). 1254

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B

A ., .*l‘.. I a.

01

02

Figure 1. Results of AG-1 immunoprecipitation of a human platelet lysate enriched in the plasma membrane fraction. The immunoprecipitate was electrophoresed in 5-15% SDS-polyacrylamide gel and transferred to nitrocellulose as described in a. The left side demonstrates molecular weight standards. The first lane demonstrates platelet membrane protein inununoprecipitated by AG-1 and stained by Amide Black 1OB. The second lane shows '251-labeled platelet &rfaCe proteins immunoprecipitated by AG-1. The third lane shows an immunoblot of a human platelet lysate enriched in the plasma membrane fraction reacted with AG-1 and identified by peroxidase conjugated goat anti-mouse IgG as described in m. This figure is representative of 4 separate experiments. Figure 2. The Mr 21,000 plgtelet surface protein immunoprecipitated by sheets containing AG-1 specifically binds [a P]GTP. Nitrocellulose transferred proteins were incubated with 50 Wi [a3'P]GTP (3000 in the Ci/mmole) alone (A) or with 50 /LC!i [a"P]GTP (3000 Ci/mmole) This molar excess of non-radioactive GTP (B). presence of a loo-fold figure is representative of 4 separate experiments.

We next activation

tested is

the

associated

protein.

Nitrocellulose

determine

if

surface

AG-1

binding

of

that

with

ligand

blots

were

Figure the

radiolabeled

incubated

2A demonstrates protein not

observed

platelet

with with

a GTP-binding

transferred GTP is

AG-l-induced

interacting

immunoprecipitates

of platelets.

[a3*P]GTP bound to

hypothesis

a low Mr G

[a3*P]GTP to protein

a broad

from

band of

band at Mr 21,000. at the

the

Mr 36,000

This band.

Figure 2B demonstrates that a loo-fold molar excess of unlabeled GTP inhibits completely the binding of [a3*P]GTP to the Mr 21,000 protein. Results of these experiments demonstrate that AG-1 immunoprecipitates specifically binds

a Mr 21,000 protein from the platelet confirms that GTP. This observation

membrane that a structural The

component of the platelet AG-1 receptor is a low Mr G protein. AG-1 receptor itself may be a low Mr G protein or, alternatively, receptor

may

coprecipitated

interact by the

physically

with

AG-1 antibody. 1255

a low Mr G protein

that

this is

Vol.

171, No. 3, 1990

These data extracellular a potent

BIOCHEMICAL

suggest that a low Mr G protein signal transduction in intact

stimulator

functional PIP,-specific

of platelet

hypothesis

may be involved human platelets.

aggregation

a low Mr G protein

that

this

a low Mr G protein presently.

ligand,

in AG-1 is These

and secretion.

structure

Previous

work

is

receptor,

the

activation

of the

has demonstrated

platelet

Mr 24,000

directly

physiological well as the signalling

This

information

interacts

is

that

not

the

may allow

with

AG-1 binding

is not

one to

PLC.

known

light chain of GpIIb between the platelet

CD9 antigen

of

the

of PIPz-specific

Further work may determine the established (3). of the AG-1 receptor and define its interactions G proteins.

activation

with

AG-1 receptor

of human platelets is not the The relationship of GPIb (1). and the

consistent

or its

in effecting

The molecular

subunit receptor

RESEARCH COMMUNICATIONS

responses are the result of receptor-mediated PLC (3). Our data demonstrating that AG-1

immunoprecipitates

protein

AND BIOPHYSICAL

or the AG-1

fully

structural with

beta

identity

platelet

determine

low Mr

the

importance of low Mr G proteins in human platelets, precise molecular interactions responsible for their

as

capabilities. ACKNOWLEDGMENTS

This work was supported by NIH grants HL02311 (MHK) and HL32853 (JIM), and an award from the Biomedical Research Support Group of Baylor College of Medicine (MHK). The authors thank Deanna Golden for the preparation of this manuscript.

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Miller, J-L., 68,743-751.

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The monoclonal antibody AG-1, a potent stimulator of human platelets, interacts with a low molecular weight GTP-binding protein.

AG-1 is a monoclonal antibody that binds to human platelets and causes aggregation and secretion. Previous work has established that these responses r...
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