Dataset Information

Skelemin in integrin ?(IIb)?(3) mediated cell spreading.

ABSTRACT: Skelemin, a myosin-associated protein in skeletal muscle, has been demonstrated to interact with integrin ?(IIb)?(3) in nonmuscle cells during initial stages of cell spreading. The significance of this interaction and the role of skelemin in integrin signaling and cytoskeletal reorganization were investigated in this study. We established a series of Chinese hamster ovary cell lines expressing wild-type or mutant ?(IIb)?(3) receptors in which skelemin binding residues at the membrane proximal region of integrin tails were mutated to alanine. Most cells displayed unimpaired adhesive capacity and spreading on immobilized fibrinogen at the early stages of cell spreading. In addition, they formed normal focal adhesions and stress fibers with no indication of impaired cell spreading. R995A/R997A/L1000A, H722A, and K716A exhibited the greatest cell spreading, which was associated with enhanced p-Src activation but was independent of FAK activation. Transfection of the cells with GFP-skelemin, containing only the C2 integrin binding domain, caused wild-type cells to round up, but had no effect on R995A/R997A/L1000A, H722A, and K716A cell spreading. Furthermore, the protrusions of the leading edge of K716A cells showed strong colocalization of talin with ?(IIb)?(3) which was associated with a loss in skelemin binding. Thus, we propose that during early stages of cell spreading, skelemin exerts contractile force on cell spreading and modulates the attachment of cytoskeletal proteins and Src to integrin clusters.

SUBMITTER: Li X

PROVIDER: S-EPMC3560429 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Skelemin in integrin α(IIb)β(3) mediated cell spreading.

Li Xinlei X Liu Yongqing Y Haas Thomas A TA

Biochemistry 20130115 4

Skelemin, a myosin-associated protein in skeletal muscle, has been demonstrated to interact with integrin α(IIb)β(3) in nonmuscle cells during initial stages of cell spreading. The significance of this interaction and the role of skelemin in integrin signaling and cytoskeletal reorganization were investigated in this study. We established a series of Chinese hamster ovary cell lines expressing wild-type or mutant α(IIb)β(3) receptors in which skelemin binding residues at the membrane proximal re ...[more]

PMID: 23273221

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Project description:Binding sites on glycoprotein (GP) IIb/IIIa exposed by 0.5 unit/ml alpha-thrombin are insensitive to prostaglandin I2 (PGI2), in contrast with sites exposed by ADP or platelet-activating factor. Here we show that the thrombin receptor agonist peptide (TRAP) (SFLLRN; 15 microM) opens almost the same number of GPIIb/IIIa molecules as 0.5 unit/ml alpha-thrombin (64840 +/- 8920 compared with 81050 +/- 6030 molecules of fibronectin bound/platelet), but these sites rapidly close on addition of PGI2. To investigate whether alpha-thrombin and TRAP initiate different signalling pathways, we measured phospholipase C (PLC)-mediated control of GPIIb/IIIa and its sensitivity to cyclic AMP. Optimal concentrations of alpha-thrombin and TRAP activated PLC maximally, but TRAP induced only about 50% protein kinase C PKC) activation after 10 min stimulation compared with alpha-thrombin. These concentrations also suppressed PGI2-induced cyclic AMP accumulation, with alpha-thrombin inducing complete inhibition and TRAP about 10% less. Direct activation of PKC by phorbol 12-myristate 13-acetate confirmed earlier observations that PGI2-induced cyclic AMP accumulation is partly inhibited via PKC. Applying different concentration of alpha-thrombin, TRAP or a combination of alpha-thrombin and the thrombin receptor inhibitory peptide (TRIP) (Mpr-F-Cha-Cha-RKPNDK-NH2; 800 microM) (Mpr, 3-mercaptopropionic acid; Cha, cyclohexylalanine), we show that the different means of stimulating the thrombin receptor all suppressed PGI2-induced cyclic AMP accumulation via (i) activation of PKC and (ii) activation of the heterotrimeric G-protein, Gi. We conclude that complete inhibition of cyclic AMP accumulation requires activation of both PKC and Gi, as observed with 0.5 unit/ml alpha-thrombin. Although TRAP almost fully exposes GPIIb/IIIa, its activation of PKC is incomplete, enabling PGI2 to raise cyclic AMP concentration from 1.4 +/- 0.7 to 4.1 +/- 1.3 nmol/10(11) platelets (P < 0.005) which is sufficient to close exposed GPIIb/IIIa molecules.

Project description:The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.

Dataset Information

Skelemin in integrin ?(IIb)?(3) mediated cell spreading.

Publications

Skelemin in integrin α(IIb)β(3) mediated cell spreading.

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