Integrin structure and signaling

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1 Integrin structure and signaling Ramray Bhat

2 History -1 Richard Hynes begins his stint in Michael Stoker s laboratory to look for cell surface changes in PyMT virus transformed cells Concept of receptors ; demonstration using lectins of altered cell surfaces; Singer Nicholson model Tryst with Iodine Discovery of Large External Transformation-Sensitive Protein (in newly discovered SDS gels(!) Not a detergent soluble integral membrane protein but fibrillar localization. ( when added affects not growth but adhesion and morphology) Hypothesizing and showing a link between cytoskeleton and ECM (change in assembly of one with changing the other) (Hynes and Destree, Cell, 1976; Ali and Hynes, 1977)

3 History-2 Use of proteolytic fragmentation studies to test what is the minimal essential part of fibronectin to bind to cells: RGDS JG-22 and CSAT two antibodies that blocked myoblast adhesion to matrix coated surfaces Damsky and Wen-Tien showed ICC that lined the antibody staining with actin and fibronectin IP of CSAT complex and affinity chromatography with elution led to a transmembrane protein with interesting properties: integrin

4 Introduction 1. Integrins are heterodimers of non-covalently associated α and β subunits 2. In vertebrates, there are 18 α and 8 β subunits that can assemble into 24 different receptors with different binding properties and different tissue distribution (Hynes 2002; Barczyk et al. 2010). 3. The α and β subunits are multidomain with flexible linkers between them. 4. Each subunit has a single membrane-spanning helix and, usually, a short unstructured cytoplasmic tail. 5. The breakthrough crystal structure of αvβ3 (Xiong et al. 2001)

5 Subdomains Campbell and Humphries, CSHP, 2011 All crystallized structures are in a bent conformation (ligand binding site pointing to membrane surface) Structural studies of intact domains reveals a more extended conformation switchblade versus deadbolt

6 α leg ectodomain 1. a 7-bladed β-propeller, Nine of the 18 integrin α chains also have α-i domain (200 aa), between blades 2 and 3 of β-propeller. The domain is vwa domain-like with five β-sheets surrounded by seven α helices. Unlike the other four relatively rigid α- leg domains, α-i domains show conformational changes within the domain important for regulating binding affinity. 2. The last three or four blades of the β-propeller contain calcium ion binding sites on the lower side. Ca ++ ion binding influences ligand binding. 3. The thigh and calf domains have similar, IgG-like, β-sandwich folds. 4.Two main regions of interdomain flexibility: between the β-propeller and the thigh, the genu between thigh and the calf-1. The α-subunit genu is located close to the similar bend in the β subunit. Campbell and Humphries, CSHP, 2011

7 β Leg Ectodomains The β-leg has 7 domains with flexible interconnections. A β-i domain in a hybrid domain, in a plexin-semaphorin-integrin (PSI) domain; 4 cysteine-rich epidermal growth factor (EGF) modules and a β-tail domain. The hybrid domain in the upper β-leg has a β- sandwich construction. The β-i domain is homologous to the α-i domain. The small PSI domain has an α/β fold. Each EGF module has an even number of eight cysteines, bonded in a C1-C5, C2-C4, C3-C6, and C7-C8 pattern except for EGF1, which lacks the C2-C4 disulfide. The β-t has an α+ β fold Campbell and Humphries, CSHP, 2011

8 The β-leg potentially more flexible than the α-leg. The EGF domain region is plastic, especially between EGF1 and EGF2, the β knee, and at the PSI/hybrid and hybrid/i-egf1 junctions. conformational changes occurring in the β-i/hybrid region. A transition from a closed to an open conformation of the β -I domain has been observed when the β-i α7-helix moves toward the hybrid domain (Xiao et al. 2004). The connecting rod-like motion of the α7-helix causes the hybrid domain to swing-out by 60 o Intrinsic ligand Glu Campbell and Humphries, CSHP, 2011

9 D723-R995 electrostatic interaction Association is consisting with the resting state of the receptor Campbell and Humphries, CSHP, 2011 β tail has two NPXY domains. Talin (PTB domain) binds the membrane proximal NPXY domain. Kindlin binds the next NPXY domain Association of talin with Beta cytoplasmic tail breaks the electrostatic interaction between α and β

10 Ligand binding to Beta propeller (alpha leg) and BetaI domain (Beta leg) or to αi domain of αleg Conformational change in receptor allowing for exposure of cytoplasmic domains to cognate binding proteins Bending-extending activation Bent topology moves external binding pocket towards cell surface away from external milieu. Breaking the bond between two legs results in extended configuration