A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3

Thomas J. Kunicki, Douglas S. Annis, Yang Jia Deng, Joseph C Loftus, Sanford J. Shattil

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The Arg-Gly-Asp (RGD) sequence within the third complementarity- determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin α(IIb)β3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V(H) domain + C(γ)1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified α(IIb)β3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 μm Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant α(IIb)β3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin α(IIb)β3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of α(IIb)β3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Original languageEnglish (US)
Pages (from-to)20315-20321
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number34
DOIs
StatePublished - 1996
Externally publishedYes

Fingerprint

Platelets
Integrins
Blood Platelets
Modulation
Ligands
Molecules
Conformations
Gels
Complementarity Determining Regions
Spodoptera
Baculoviridae
Divalent Cations
Coinfection
Amino Acid Sequence
Complementary DNA
Chemical activation
Cells
Amino Acids
Cell Line

ASJC Scopus subject areas

  • Biochemistry

Cite this

A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3 . / Kunicki, Thomas J.; Annis, Douglas S.; Deng, Yang Jia; Loftus, Joseph C; Shattil, Sanford J.

In: Journal of Biological Chemistry, Vol. 271, No. 34, 1996, p. 20315-20321.

Research output: Contribution to journalArticle

Kunicki, Thomas J. ; Annis, Douglas S. ; Deng, Yang Jia ; Loftus, Joseph C ; Shattil, Sanford J. / A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3 In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 34. pp. 20315-20321.
@article{f7a9e5c2b4774c088758700a6bad16fa,
title = "A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3",
abstract = "The Arg-Gly-Asp (RGD) sequence within the third complementarity- determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin α(IIb)β3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V(H) domain + C(γ)1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified α(IIb)β3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17{\%} in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40{\%} in the presence of 10 μm Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant α(IIb)β3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin α(IIb)β3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of α(IIb)β3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.",
author = "Kunicki, {Thomas J.} and Annis, {Douglas S.} and Deng, {Yang Jia} and Loftus, {Joseph C} and Shattil, {Sanford J.}",
year = "1996",
doi = "10.1074/jbc.271.34.20315",
language = "English (US)",
volume = "271",
pages = "20315--20321",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "34",

}

TY - JOUR

T1 - A molecular basis for affinity modulation of Fab ligand binding to integrin α(IIb)β3

AU - Kunicki, Thomas J.

AU - Annis, Douglas S.

AU - Deng, Yang Jia

AU - Loftus, Joseph C

AU - Shattil, Sanford J.

PY - 1996

Y1 - 1996

N2 - The Arg-Gly-Asp (RGD) sequence within the third complementarity- determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin α(IIb)β3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V(H) domain + C(γ)1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified α(IIb)β3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 μm Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant α(IIb)β3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin α(IIb)β3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of α(IIb)β3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

AB - The Arg-Gly-Asp (RGD) sequence within the third complementarity- determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin α(IIb)β3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (V(H) domain + C(γ)1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 κ chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified α(IIb)β3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1.1 Fab molecules bound per platelet was 17% in the presence of 1 mM Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 μm Mn2+. The ratio of Fab molecules bound after versus before activation (mean ± S.D.; n = 3) was: for AP7.3, 9.8 ± 0.6; for PAC1.1, 8.8 ± 0.3; and for AP7, 1.4 ± 0.2. In addition, AP7 bound to the stably expressed integrin mutant α(IIb)β3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin α(IIb)β3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of α(IIb)β3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

UR - http://www.scopus.com/inward/record.url?scp=0029833707&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029833707&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.34.20315

DO - 10.1074/jbc.271.34.20315

M3 - Article

C2 - 8702765

AN - SCOPUS:0029833707

VL - 271

SP - 20315

EP - 20321

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -