A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3

Hirokazu Kashiwagi, Yoshiaki Tomiyama, Seiji Tadokoro, Shigenori Honda, Masamichi Shiraga, Hajime Mizutani, Makoto Handa, Yoshiyuki Kurata, Yuji Matsuzawa, Sanford J. Shattil

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, α(IIb)β3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of α(IIb)β3. One clone (AM-1) expressed constitutively active α(IIb)β3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiα(IIb)β3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated α(v)β3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated α(IIb)β3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to α(IIb)β3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Original languageEnglish
Pages (from-to)2559-2568
Number of pages10
JournalBlood
Volume93
Issue number8
Publication statusPublished - 1999 Apr 15
Externally publishedYes

Fingerprint

Integrins
Fibrinogen
Cysteine
Mutation
Chemical activation
Ligands
Platelets
Tail
Glycosylation
Cell Aggregation
Phosphorylation
Asparagine
Platelet Activation
Amino Acid Substitution
Cricetulus
Protein-Tyrosine Kinases
Sequence Analysis
Tyrosine
Ovary
Substitution reactions

ASJC Scopus subject areas

  • Hematology

Cite this

Kashiwagi, H., Tomiyama, Y., Tadokoro, S., Honda, S., Shiraga, M., Mizutani, H., ... Shattil, S. J. (1999). A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3. Blood, 93(8), 2559-2568.

A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3. / Kashiwagi, Hirokazu; Tomiyama, Yoshiaki; Tadokoro, Seiji; Honda, Shigenori; Shiraga, Masamichi; Mizutani, Hajime; Handa, Makoto; Kurata, Yoshiyuki; Matsuzawa, Yuji; Shattil, Sanford J.

In: Blood, Vol. 93, No. 8, 15.04.1999, p. 2559-2568.

Research output: Contribution to journalArticle

Kashiwagi, H, Tomiyama, Y, Tadokoro, S, Honda, S, Shiraga, M, Mizutani, H, Handa, M, Kurata, Y, Matsuzawa, Y & Shattil, SJ 1999, 'A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3', Blood, vol. 93, no. 8, pp. 2559-2568.
Kashiwagi H, Tomiyama Y, Tadokoro S, Honda S, Shiraga M, Mizutani H et al. A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3. Blood. 1999 Apr 15;93(8):2559-2568.
Kashiwagi, Hirokazu ; Tomiyama, Yoshiaki ; Tadokoro, Seiji ; Honda, Shigenori ; Shiraga, Masamichi ; Mizutani, Hajime ; Handa, Makoto ; Kurata, Yoshiyuki ; Matsuzawa, Yuji ; Shattil, Sanford J. / A mutation in the extracellular cysteine-rich repeat region of the β3 subunit activates integrins α(IIb)β3 andα(v)β3. In: Blood. 1999 ; Vol. 93, No. 8. pp. 2559-2568.
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