A structural basis for integrin activation by the cytoplasmic tail of the alpha IIb-subunit

Abstract

A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short alpha- and/or beta-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membrane-anchored cytoplasmic tail of the alpha(IIb)-subunit and of a mutant alpha(IIb)-cytoplasmic tail that renders platelet integrin alpha(IIb)beta(3) constitutively active. The structure of the wild-type alpha(IIb)-cytoplasmic tail reveals a "closed" conformation where the highly conserved N-terminal membrane-proximal region forms an alpha-helix followed by a turn, and the acidic C-terminal loop interacts with the N-terminal helix. The structure of the active mutant is significantly different, having an "open" conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed alpha(IIb)beta(3) activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical/mutational data and support a conformation-based "on/off switch" model for integrin activation.

Figure 1

Sequence and function of the integrin α_IIb-cytoplasmic domain. (A) Sequence alignment of various human integrin α-cytoplasmic domains with both wild-type and mutant α_IIb. The sequence identity and similarity are highlighted in decreased darkness. Residue numbers correspond to α_IIb. (B) Effect of α_IIb-cytoplasmic peptides on the binding of fibrinogen (Fg) to stimulated platelets. Human platelets in Tyrode's buffer alone (buffer), with or without 50 μM (final concentration) of nonmyristoylated α_IIb K989–E1008 (α_IIb), m-α_IIb-wt (m-α_IIb), and myristic acid (Myr) were preincubated for 30 min with 300 nM ¹²⁵I-fibrinogen. The platelets were then stimulated with either 10 μM ADP/20 μM epinephrine (shaded bars) or 50 μM thrombin receptor agonist peptide (hatched bars). After 15 min, ¹²⁵I-fibrinogen binding was measured. Incubation of nonstimulated platelets with cytoplasmic peptides or with myristic acid had no effect on fibrinogen binding (data not shown). (C) Effects of α_IIb-cytoplasmic peptides on the binding of fibrinogen to immunocaptured forms of α_IIbβ₃. Both forms of the receptor, platelet α_IIbβ₃ (shaded bars) or sr-α_IIbβ₃ (hatched bars), were immunocaptured onto immobilized monoclonal antibody AP3. The captured receptors were preincubated for 30 min with either buffer alone (buffer) or 50 μM nonmyristoylated α_IIb K989–E1008 (α_IIb). Peptide specificity was confirmed with a nonrelated acidic peptide (control peptide; GDKNADGWIEFEEL). Results are expressed as a percentage of maximal fibrinogen binding in the absence of peptides. (D) Divergent effects of myristoylated wild-type and mutant α_IIb-cytoplasmic peptides on the binding of fibrinogen to stimulated platelets. Platelets were preincubated with varying concentrations of either m-α_IIb-wt (closed circles) or m-α_IIb-mut peptides (open circles), and then ¹²⁵I-fibrinogen binding to ADP/epinephrine-stimulated platelets was measured.

Figure 2

Selected regions of two dimensional ¹H NOESY for m-α_IIb-wt and m-α_IIb-mut at 25°C and pH 5.0. (A) Portion of aliphatic region for m-α_IIb-wt, showing K994–E1005 contact. (B) Portion of aromatic region for m-α_IIb-wt, showing F993–P998 contact. (C) Region corresponding to that shown in A for m-α_IIb-mut. (D) Region corresponding to that shown in B for m-α_IIb-mut. Note that other than loss of long-range NOEs for the mutant, the mutant had fewer sequential NOEs than the wild type (see text).

Figure 3

Illustration of m-α_IIb-wt and m-α_IIb-mut structures. (A) Backbone superpositions of the 15 best m-α_IIb-wt structures. (B) Backbone ribbon diagram of minimized average m-α_IIb-wt structure in the same view as in A. (C) Backbone superpositions of m-α_IIb-mut structures. (D) Backbone ribbon diagram of minimized average m-α_IIb-mut structure in the same view as in C. The structural statistics are listed in Table 1.

Figure 4

Structural highlights of the cytoplasmic domain of α_IIb. (A) Structure of the m-α_IIb-wt showing the side chains of K994, R997, E1001, D1003, D1004, and E1005 for the salt-bridge network. A proposed Ca²⁺-binding site is also shown in the figure involving R997, E1001, D1003, and D1004, which were identified by comparing the two-dimensional ¹H total correlation spectroscopy spectra of Ca²⁺-free and Ca²⁺-bound forms (Ca²⁺ was added up to 5-fold). The binding of Ca²⁺ to the α_IIb-tail has been shown by biochemical studies (12, 27). (B) Backbone overlay of m-α_IIb-wt (pink) and m-α_IIb-mut (yellow), showing the structural difference. The C-terminal loop of m-α_IIb-wt folds back to interact with the N-terminal helix, whereas in the mutant, the C-terminal part is highly flexible and makes no interactions with the helix. The C-terminal part (D1003–E1008) of the m-α_IIb-mut is disordered.

Figure 5

Model for the regulation of ligand binding to α_IIbβ₃. In resting platelets, α_IIbβ₃ is maintained in an inactive/resting state (a) by a negative regulator (− circle). The negative regulator may be the interaction between the α/β-cytoplasmic tails and/or an intracellular protein. The α-cytoplasmic tail adopts a closed conformation with its N terminus (α-N) interacting with its acidic C terminus (α-C). Platelet stimulation (Agonist) leads to the association of intracellular constituents (positive regulator, + circle) with the cytoplasmic domain of α_IIbβ₃, generating an “activating conformational signal” (b) such that fibrinogen binds to the receptor (c). m-α_IIb-wt converts the b state back into the inactive a state by forming an intermediate d, an a → b → d → a pathway. The N-terminal α_IIb-cytoplasmic peptide perturbs the α/β-complex shown in a, generating an intermediate e, which shifts the equilibrium to c state for binding of fibrinogen, the a → e → c pathway. Selective mutations (see text) in either the N or C terminus of α_IIb or β₃ can also lead directly to the expression of a constitutively active receptor (f), bypassing the a → b → c pathway.