https://www.auburn.edu/academic/classes/biol/6190/refs/L04/1-s2.0-S0092867402009716-main.pdf

Integrins: Bidirectional, Allosteric Signaling Machines

Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes. This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them, and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.

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Cell migration: integrating signals from front to back.

Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed polyproline II-type helices. Much progress has been made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen—the prototypical collagen fibril—has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology.

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Collagen Structure and Stability

The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.

Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with an Arg-Gly-Asp Ligand

Integrins are cell adhesion molecules that mediate cell-cell, cell– extracellular matrix, and cell-pathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 ˚ A couple to conformational change in ligand-binding sites and are linked to changes in α and β subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.

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Structural Basis of Integrin Regulation and Signaling

Odumosu and Wilson1 have reported the unexpected observation that a significant proportion of female guinea-pigs were able to survive for an unusually long period (in some cases perhaps indefinitely) on a diet free of ascorbic acid, implying that these animals could synthesize the vitamin endogenously. The literature on experimental scurvy in guinea-pigs contains, as far as we are aware, no past studies on the mortality of guinea-pigs deprived of an exogenous source of ascorbic acid in relation to sex. In experiments the animals were killed before they reached the point where death from scurvy might be expected, and the sex of animals was either not stated or male only. Nevertheless it has been assumed that both male and female guinea-pigs require an exogenous supply of ascorbic acid and that administration of an ascorbic acid-free diet would lead to the death of all animals, irrespective of sex, within 3–4 weeks. In view of the importance of the observation of Odumosu and Wilson1 in respect of the nature of the lesion in ascorbic acid metabolism in guinea-pigs and as it might reflect on the requirements of humans for an exogenous supply of the vitamin, we felt it necessary to verify their finding.

Mortality Rate in Male and Female Guinea-pigs on a Scorbutogenic Diet

Freshly isolated bone cells from embryonic chick calvariae were found to attach to lilms of native monomeric type I or II collagen, to gels of native type I collagen and, to a lesser extent, to air-dried films of these collagens, in a libronectin-independent manner, and with no requirement for collagen synthesis. Adhesion to type III collagen was relatively poor. Films or native monomeric type I or II collagen were preferred to the corresponding air-dried films as substrata for proliferation. Cis-hydroxyproline inhibited growth upon both plastic and type I and type II collagenous substrata, but this effect could largely be overcome by providing bone cell-conditioned medium, suggesting the possibility of a requirement in the growth process for synthesis of a collagen other than type I, the major form in bone matrix.

Interaction of Embryonic Chick Calvarial Bone Cells with Collagen Substrata; Attachment Characteristics and Growth Behaviour

Collagen type VI in native (undenatured or triple-helical) form has been shown, like collagen types I-V, alpha 1(I) trimer and alpha 2(I) trimer, to possess platelet reactivity provided that essential quaternary structural needs are first satisfied. Thus platelet aggregation was induced by the collagenous domain of collagen type VI, isolated free of the non-collagenous elements, when this entity was presented to platelets in fibrillar form. This implies that platelet recognition sites in collagen type VI are located in the collagenous sequence of the molecule. Aggregation of platelets was also induced, although a higher concentration was required, by the intact, "parent" collagen following its polymerisation by random association of molecules with the aid of a cross-linking agent (glutaraldehyde) to yield an amorphous polymer. This permits the suggestion that the more ordered molecular assembly of collagen type VI thought to occur in vivo, to yield a microfibrillar form, is likely to be associated with significant platelet reactivity. Our results support the notion that any collagenous species may be reactive towards platelets provided that essential tertiary and quaternary structural requirements are met and in this sense, therefore, they favour more the idea of multiple platelet-reactive sites in collagen of relatively low structural specificity and low affinity.

The Platelet Reactivity of Collagen Type VI

Micromolar Ca2+ Concentrations are Essential for Mg2+-dependent Binding of Collagen by the Integrin α2β1 in Human Platelets

The crystal structure of a collagen peptide in complex with integrin α2 I-domain is provided. Coordinates for the crystal structure are useful in designing novel molecules that can be tested for binding to the receptor and other I-domains and preferably ability to inhibit I-domain binding to ligand, and I-domain function. Regions of I-domains that undergo conformation change upon ligand binding are also identified and provided as targets for binding molecules such as antibodies. Molecules that inhibit the function of polypeptides comprising I-domains are of therapeutic potential in a number of diseases and disorders.

Michael J. Barnes

Papers

Mortality Rate in Male and Female Guinea-pigs on a Scorbutogenic Diet

Interaction of Embryonic Chick Calvarial Bone Cells with Collagen Substrata; Attachment Characteristics and Growth Behaviour

The Platelet Reactivity of Collagen Type VI

Micromolar Ca2+ Concentrations are Essential for Mg2+-dependent Binding of Collagen by the Integrin α2β1 in Human Platelets

Receptor/peptide crystal structure for identification of inhibitors