Structure of the integrin alphaIIb transmembrane segment

Integrin cell-adhesion receptors transduce signals bidirectionally across the plasma membrane via the single-pass transmembrane segments of each alpha and beta subunit. While the beta3 transmembrane segment consists of a linear 29-residue alpha-helix, the structure of the alphaIIb transmembrane segment reveals a linear 24-residue alpha-helix (Ile-966 -Lys-989) followed by a backbone reversal that packs Phe-992-Phe-993 against the transmembrane helix. The length of the alphaIIb transmembrane helix implies the absence of a significant transmembrane helix tilt in contrast to its partnering beta3 subunit. Sequence alignment shows Gly-991-Phe-993 to be fully conserved among all 18 human integrin alpha subunits, suggesting that their unusual structural motif is prototypical for integrin alpha subunits. The alphaIIb transmembrane structure demonstrates a level of complexity within the membrane that is beyond simple transmembrane helices and forms the structural basis for assessing the extent of structural and topological rearrangements upon alphaIIb-beta3 association, i.e. integrin transmembrane signaling.     

A naturally occurring extracellular alpha-beta clasp contributes to stabilization of beta3 integrins in a bent, resting conformation

Control of alphaIIb beta3 and alphav beta3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin alpha and beta subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or "off" conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of alphav beta3. In this bent structure, a segment of alphav (301-308) and beta3 (560-567) are juxtaposed. Here we provide evidence that these regions of alphav/alphaIIb and beta3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the alphaIIb and beta3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three beta3 LIBS antibodies. Mutation of the clasp region of alphav or beta3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the alphav beta3 structure yield a refined model for the alphav beta3 clasp and provide plausible explanations for the effects of the activating mutations.     

Activation of platelet alphaIIbbeta3 by an exogenous peptide corresponding to the transmembrane domain of alphaIIb

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.     

Transmembrane helices that form two opposite homodimeric interactions: an asparagine scan study of alphaM and beta2 integrins

Integrins are alpha/beta heterodimers, but recent in vitro and in vivo experiments also suggest an ability to associate through their transmembrane domains to form homomeric interactions. While the results of some in vitro experiments are consistent with an interaction mediated by a GxxxG-like motif, homo-oligomers observed after in vivo cross-linking are consistent with an almost opposite helix-helix interface. We have shown recently that both models of interaction are compatible with evolutionary conservation data, and we predicted that the alpha-helices in both models would have a similar rotational orientation. Herein, we have tested our prediction using in vitro asparagine scan of five consecutive residues along the GxxxG-like motif of the transmembrane domain of alpha and beta integrins, alphaM and beta2. We show that Asn-mediated dimerization occurs twice for every turn of the helix, consistent with two almost opposite forms of interaction as suggested previously for alphaIIb and beta3 transmembrane domains. The orientational parameters helix tilt and rotational orientation of each of these two Asn-stabilized dimers were measured by site-specific infrared dichroism (SSID) in model lipid bilayers and were found to be consistent with our predicted computational models. Our results highlight an intrinsic tendency for integrin transmembrane alpha-helices to form two opposite types of homomeric interaction in addition to their heteromeric interactions and suggest that integrins may form complex and specific networks at the transmembrane domain during function.     

Collagen XVII promotes integrin-mediated squamous cell carcinoma transmigration--a novel role for alphaIIb integrin and tirofiban

The expression of collagen XVII (BP180), a transmembrane hemidesmosomal component, is upregulated in invasive areas of epithelial tumors. The collagenous ectodomain of collagen XVII is cleaved from the plasma membrane of keratinocytes and malignant epithelial cells. The released ectodomain is predicted to regulate cell detachment, differentiation, and motility. We report that the cell adhesion domain of collagen XVII, Col15, is able to chemotactically attract invasive HSC-3 SCC cells but not keratinocytes. Analysis of integrin expression revealed that HSC-3 cells, unlike keratinocytes, express alphaIIb integrin, a platelet-specific fibrinogen receptor. We show that this novel chemotactic function is mediated by the known Col15-binding integrins alpha5beta1 and alphav and the platelet integrin alphaIIb. Moreover, we report that tirofiban, a FDA-proved alphaIIb integrin-blocking drug widely used for the therapy of acute coronary ischaemic syndrome and the prevention of thrombotic complications, inhibits the Col15 chemotaxis of HSC-3 carcinoma cells. Together, these data demonstrate a novel interaction between collagen XVII and alphaIIb integrin and also suggest a possibility to use tirofiban to inhibit the invasion and progression of alphaIIb expressing SCC tumors.     

Mechanistic insights from a refined three-dimensional model of integrin alphaIIbbeta3

The integrin alpha(IIb)beta(3) plays an important role in platelet function, and abnormalities of this protein result in a serious bleeding disorder, known as Glanzmann thrombasthenia. Although crystallographic data exist for the related integrin alpha(V)beta(3), to date, there are no high resolution structures of integrin alpha(IIb)beta(3) available in the literature. Therefore, it is still unclear how specific elements of the alpha(IIb) subunit contribute to integrin alpha(IIb)beta(3) function. Here we describe a refined model of the alpha(IIb) N-terminal portion of integrin alpha(IIb)beta(3) obtained by using the alpha(V)beta(3) template combined with a new method for predicting the conformations of the unique alpha(IIb) loop regions comprising residues 71-85, 114-125, and 148-164. The refined model was probed based on a structural prediction that differentiates it from standard homology models: specifically, that Lys-118 of alpha(IIb) contacts Glu-171 of beta(3). To test this hypothesis experimentally, the mutant integrin chains alpha(IIb) K118C and beta(3) E171C were cotransfected into HEK 293 cells. We show that the cells expressed the mutants alpha(IIb)beta(3) on their surface as a disulfide-linked dimer, supporting the close proximity between alpha(IIb) Lys-118 and beta(3) Glu-171 predicted from the refined model. This validated model provides a specific structural context for the analysis and interpretation of structure-function relations of integrin alpha(IIb)beta(3). In addition, it suggests mechanistic hypotheses pertaining to both naturally occurring mutations responsible for Glanzmann thrombasthenia and to point mutations that affect ligand binding.     

Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.     

Structures of membrane proteins

Summary The possible conformations of integral membrane proteins are restricted by the nature of their environment. In order to satisfy the requirement of maximum hydrogen bonding, those protions of the polypeptide chain which are in contact with lipid hydrocarbon must be organized into regions of regular secondary structure. As possible models of the intramembranous regions of integral membrane proteins, three types of regular structues are discussed. Two, the alpha helix and the beta-pleated sheet, are regularly occurring structural features of soluble proteins. The third is a newly proposed class of conformations called beta helices. These helices have unique features which make them particularly well-suited to the lipid bilayer environment. The central segment of the membrane-spanning protein glycophorin can be arranged into a beta helix with a hydrophobic exterior and a polar interior containing charged amino-acid side chains. Such structures could function as transmembrane ion channels. A model of the activation process based on a hypothetical equilibrium between alpha and beta helical forms of a transmembrane protein is presented. The model can accurately reproduce the kinetics and voltage dependence of the channels in nerve.     

Mass spectrometric based mapping of the disulfide bonding patterns of integrin alpha chains

Integrins are one of the major mediators of cellular adherence. Structurally the component alpha and beta chains are characterized by extensive intrachain disulfide bonding. The assignment of these bonds is currently based on homology with the chains of the integrin alphaIIbbeta3. However, recent crystallographic analysis of the soluble alphaVbeta3 construct indicates that the alphaV chain displays bonding patterns different from those predicted for alphaIIb. In an effort to define the disulfide bonding patterns in integrins, we have used mass spectrometric based approaches to map the human alpha3, alpha5, alphaV, and alphaIIb. The results indicate that there are differences in the disulfide patterns of the alpha chains. These do not correlate with the integrin capacity to bind ligands as all integrins used in the present study displayed functional activity. The differences were observed in the bonding patterns linking the heavy (H) and light (L) components of the of the alpha chains. It was also possible to assign the location in alpha5 of an additional disulfide bond involving a pair of cysteines not present in alphaV or alphaIIb. This second bond between the H and L chains of alpha5 has not been previously described. These results indicate that not all integrin species display the same disulfide bonding patterns. They also highlight the need for caution in the use of assignments based on sequence homology.     

Conformations and pharmacophores of cyclic RGD containing peptides which selectively bind integrin alpha(v)beta3.

This paper reports a detailed conformational characterization in solution by 1H-NMR in H2O and DMSO-d6 and molecular modeling simulations of cyclic peptides containing the RGDDV pharmacophore and the RGDY(Me)R pharmacophore. These two pentapeptide sequences when properly constrained in cyclic peptides are low to sub-nanomolar inhibitors of integrin alpha(v)beta3. The peptides containing the RGDDY(Me)R sequence bind potently to integrin alphaIIb3 as well. The conformations found in H2O and in DMSO-d6 solutions are valuable for the design of peptidomimetics of these two pharmacophores. The structure-activity relationships of the RGDDV and RGDY(Me)R pharmacophores within cyclic peptides are discussed. Specifically, the orientation of surface-accessible chemical features on the ligand, such as hydrophobic, positive and negative ionizable groups, which are considered to be responsible for the desired biological activity, is focused on.     

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.     

Integrin structures and conformational signaling

Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.     

Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET

Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using F?rster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.     

Structural insight into the interaction between platelet integrin alphaIIbbeta3 and cytoskeletal protein skelemin

Skelemin is a large cytoskeletal protein critical for cell morphology. Previous studies have suggested that its two-tandem immunoglobulin C2-like repeats (SkIgC4 and SkIgC5) are involved in binding to integrin beta3 cytoplasmic tail (CT), providing a mechanism for skelemin to regulate integrin-mediated signaling and cell spreading. Using NMR spectroscopy, we have studied the molecular details of the skelemin IgC45 interaction with the cytoplasmic face of integrin alphaIIbbeta3. Here, we show that skelemin IgC45 domains form a complex not only with integrin beta3 CT but also, surprisingly, with the integrin alphaIIb CT. Chemical shift mapping experiments demonstrate that both membrane-proximal regions of alphaIIb and beta3 CTs are involved in binding to skelemin. NMR structural determinations, combined with homology modeling, revealed that SkIgC4 and SkIgC5 both exhibited a conserved Ig-fold and both repeats were required for effective binding to and attenuation of alphaIIbbeta3 cytoplasmic complex. These data provide the first molecular insight into how skelemin may interact with integrins and regulate integrin-mediated signaling and cell spreading.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.     

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.     

A coiled-coil structure of the alphaIIbbeta3 integrin transmembrane and cytoplasmic domains in its resting state

One of the hallmark features of the integrin receptors is the ability to transmit signals bidirectionally through the cell membrane. The transmembrane integrin domains are pivotal to the signaling events. An understanding of the signaling mechanism requires structural information. Here, we report a structural model of the transmembrane and part of the cytosolic domains of the alphaIIbbeta3 integrin in its resting state. The model was obtained computationally by a restrained conformational search of helix-helix interactions. It agrees with one published NMR structure of the cytoplasmic complex and can put many experimental findings on structural grounds. According to our model, integrins form an intricately designed coiled-coil structure in the resting state. The conserved Glycophorin A (GpA)-like sequence motif of the alpha, but not the beta, subunit, is in the interface of this model. Based on our calculations and other data, a signaling mechanism that involves a transient GpA-like structure is proposed.     

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.     

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.