Petri Nykvist Integrins As Cellular Receptors for Fibril-Forming And
Total Page：16
File Type：pdf, Size：1020Kb
Copyright © , by University of Jyväskylä ABSTRACT Nykvist, Petri Integrins as cellular receptors for fibril-forming and transmembrane collagens Jyväskylä: University of Jyväskylä, 2004, 127 p. (Jyväskylä Studies in Biological and Environmental Science, ISSN 1456-9701; 137) ISBN 951-39-1773-8 Yhteenveto: Integriinit reseptoreina fibrillaarisille ja transmembraanisille kollageeneille Diss. The two integrin-type collagen receptors α1β1 and α2β1 integrins are structurally very similar. However, cells can concomitantly express both receptors and it has been shown that these collagen receptor integrins have distinct signaling functions, and their binding to collagen may lead to opposite cellular responses. In this study, fibrillar collagen types, I, II, III, and V, and network like structure forming collagen type IV tested were recognized by both integrins at least at the αI domain level. The αI domain recognition does not always lead for cell spreading behavior. In addition transmembrane collagen type XIII was studied. CHO-α1β1 cells could spread on recombinant human collagen type XIII, unlike CHO-α2β1 cells. This finding was supported by αI domain binding studies. The results indicate, that α1β1 and α2β1 integrins do have different ligand binding specificities and distinct collagen recognition mechanisms. A common structural feature in the collagen binding αI domains is the presence of an extra helix, named helix αC. A αC helix deletion reduced affinity for collagen type I when compared to wild-type α2I domain, which indicated the importance of helix αC in collagen type I binding. Further, point mutations in amino acids Asp219, Asp259, Asp292 and Glu299 resulted in weakened affinity for collagen type I. Cells expressing double mutated α2Asp219/Asp292 integrin subunit showed remarkably slower spreading on collagen type I, while spreading on collagen type IV was not affected. The data indicated that α2I domain binds to collagen type I with a different mechanism than binding to collagen type IV. In tissues collagen monomers form large fibrils immediately after they have been released from cells. The α2I domain binding data indicate that fibril formation affects the binding constant and at the same time reduces the number of putative binding sites available to the integrins. However, integrin α2β1 could still mediate spreading on fibrillar collagen and the contraction of floating collagen gels. The α1β1 cells could neither adequately spread on collagen fibrils nor mediate the contraction of collagen gels. These findings indicate that α2β1 integrin is a functional cellular receptor for collagen fibrils, while α1β1 integrin may only bind collagen monomers effectively. Type XVII collagen, containing large collagenous domain called COL15, is a keratinocyte protein in hemidesmosomes or as soluable form in extracellular space. Assays showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Instead, the spreading on denatureted COL15 was mediated by α5β1 and αVβ1 integrins and inhibited by KGD-containing synthetic peptides. This suggests that the COL15 domain of collagen type XVII represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface it may support keratinocyte spreading and migration. Key words: Collagen fibrils; collagen monomers; integrin-type collagen receptors; transmembrane collagens. P. Nykvist, University of Jyväskylä, Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland Author’s address Petri Nykvist Department of Biological and Environmental Science P.O. Box 35 University of Jyväskylä FIN-40014 University of Jyväskylä Finland E-mail:[email protected] Supervisors Professor Jyrki Heino, M. D., Ph. D. Department of Biological and Environmental Science P.O. Box 35 University of Jyväskylä FIN-40014 University of Jyväskylä Finland Docent Jarmo Käpylä, Ph. D. Department of Biological and Environmental Science P.O. Box 35 University of Jyväskylä FIN-40014 University of Jyväskylä Finland Reviewers Docent Vuokko Kovanen, Ph. D. Department of Health Sciences P.O. Box 35 University of Jyväskylä FIN-40014 University of Jyväskylä Finland Docent Anna-Marja Säämänen, Ph. D. Department of Medical Biochemistry and Molecular Biology University of Turku FIN-20014 University of Turku Finland Opponent Docent Johanna Myllyharju, Ph. D. Department of Medical Biochemistry and Molecular Biology University of Oulu P.O. Box 5000 FIN-90014 University of Oulu Finland CONTENTS ABSTRACT LIST OF ORIGINAL PUBLICATIONS ABBREVIATIONS 1 INTRODUCTION ............................................................................................. 11 2 REVIEW OF LITERATURE.............................................................................. 13 2.1 Family of collagens................................................................................ 13 2.1.1 Fibrillar collagen ...................................................................... 14 2.1.1.1 Synthesis of fibril-forming collagens ..................... 14 2.1.1.2 Monomeric family members in fibrils ................... 16 2.1.1.3 Fibrillogenesis and fibrils formed – a collagenous puzzle ............................................... 19 2.1.2 Non-fibrillar collagens............................................................. 25 2.1.2.1 Network-forming collagens - collagen IV as prototype ................................................................. 25 2.1.2.2 Transmembrane collagens ..................................... 26 2.2 Family of integrins................................................................................. 32 2.2.1 Integrins in the beginning ....................................................... 32 2.2.2 Integrins presently ................................................................... 33 2.2.3 Collagen binding integrins...................................................... 34 2.2.3.1 Integrin α1β1 - binding preference to collagen type I. ........................................................ 34 2.2.3.2 Integrin α2β1 - a primary receptor for collagen type I ........................................................ 36 2.2.3.3 Integrins α10β1 and α11β1 - novel family members .................................................................. 38 2.2.4 Structure of integrins - a handshake between the subunits..................................................................................... 43 2.2.4.1 Integrin α subunit - a ground for collagen bing .......................................................................... 45 2.2.4.1.1 The αI domain structure ..................... 46 2.2.4.1.2 Integrin-collagen interaction – a cocrystal............................................. 49 2.2.4.2 Integrin β1 subunit with βI like domain............... 52 3 AIMS OF THE STUDY..................................................................................... 54 4 MATERIALS AND METHODS ...................................................................... 55 4.1 Preparation of collagens........................................................................ 55 4.1.1 Generation of recombinant ectodomain of human collagen type XIII (I) .............................................................. 55 4.1.2 Generation of recombinant COL15 domain of human collagen type XVII (IV) ......................................................... 56 4.1.3 In vitro collagen type I fibrillogenesis (III) ........................... 56 4.2 Studies on integrin αI domain - collagen interaction (I-IV).............. 56 4.2.1 Generation of human recombinant integrin α1I and α2I domains (IV) ...................................................... 56 4.2.2 Generation of mutated α2I domains (II) .............................. 57 4.2.3 Generation of human recombinant α10I domain as MBP-fusion protein (IV) ........................................................ 58 4.2.4 Direct Europium labeling of αI domains (I, II) .................... 58 4.2.5 Binding assay for directly Europium labeled αI domains (I, II) ..................................................................... 59 4.2.6 Binding assay for αI domains as GST and MBP fusion proteins (III, IV) ................................................. 59 4.2.7 Electron microscopy to study collagen type I fibril - αI domain interaction (III) ..................................................... 60 4.2.8 IAsys and BIAcore biosensor experiments (II) .................... 60 4.2.9 Molecular modeling (II) ......................................................... 60 4.3 Studies on cell - collagen interaction (I-IV) ........................................ 61 4.3.1 Transfection of CHO cells to express α1β1 or α2β1 integrins (I-IV) ............................................................... 61 4.3.2 Generation of CHO cell lines to express mutated α2β1 integrins (II, III) ............................................................. 61 4.3.3 Cell spreading assays (I-IV) ................................................... 62 4.3.4 Collagen gel contraction assay (III) ...................................... 63 4.3.5 Lateral cell migration assay (IV) ........................................... 63 5 RESULTS.......................................................................................................... 64 5.1 Collagen binding profile of α1β1 integrin is different than α2β1 integrin (I) ........................................................................... 64 5.1.1 Methods