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Folding of the conserved domain

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Proc. Natl. Acad. Sci. USAVol. 94, pp. 3156–3161, April 1997ImmunologyFolding of the conserved domain but not of flanking regions inthe integrinb2subunit requires association with theasubunitCHICHI HUANG,CHAFEN LU, AND TIMOTHY A. SPRINGER*The Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115Contributed by Timothy A. Springer, January 3, 1997ABSTRACT We have used immunoprecipitation withmAbs to probe folding during biosynthesis of theb2integrinsubunit of lymphocyte function-associated antigen 1 (LFA-1;CD11ayCD18) before and after association with theaLsub-unit. An evolutionarily conserved region is present in theb2subunit between amino acid residues 102 and 344. mAbs to onesubregion before the conserved region, and two subregionsafter the conserved domain, immunoprecipitated both theunassociatedb*2precursor and matureaLyb2complex, sug-gesting portions of these subregions are folded before asso-ciation withaL. An activating mAb to the C-terminal cysteine-rich region, KIM127, preferentially bound to the unassociatedbsubunit, suggesting that it may bind to an epitope that is inanabinterface in unactivated LFA-1. By contrast, mAbs tofive different epitopes in the conserved region did not reactwith unassociatedb*2precursor, suggesting that this regionfolds afteraLassociation and is intimately associated with theaLsubunit in theaLyb2complex. mAbs to two differentepitopes that involve the border between the conserved regionand the C-terminal segment, were fully or partially reactivewith theb*2precursor, suggesting that this region is partiallyfolded before association withaL. The findings suggest thatthe conserved region is a distinct folding and hence structuralunit, and is intimately associated with theasubunit.Integrins are a family of cell surface glycoproteins that mediatecell–cell and cell–substrate adhesion, extracellular matrix as-sembly, and signal transduction (1). Integrins are heterodimersconsisting of noncovalently associatedaandbsubunits. Theleukocyte integrins are restricted to leukocytes and share theb2integrin or CD18 subunit (2, 3). Theb2subunit can formfour differentabcomplexes containing theaL(CD11a) sub-unit of lymphocyte function-associated antigen 1 (LFA-1), theaM(CD11b) subunit of Mac-1, theaX(CD11c) subunit ofp150,95, and theaDsubunit ofaDyb2(4–6). The leukocyteintegrins are essential in many phases of immune and inflam-matory responses (7). Mutations in theb2integrin subunit areresponsible for leukocyte adhesion deficiency I and result inrecurring and life-threatening infections (8, 9).Much remains to be learned about the structure of integrins.An overall view is provided by electron microscopy, whichshows a globular head region that binds ligand, contains theN-terminal portions of theaandbsubunits and is connectedto the membrane by two stalks corresponding to extended,more C-terminal regions of theaandbsubunits (10). Severaldomains or subregions in integrins have been identified. TheN-terminal region of integrinasubunits contains seven re-peats of '60 aa each that have been predicted to fold into acylindrical structure with sevenb-sheets known as ab-pro-peller domain (11). An inserted (I) domain is located betweenb-sheet 2 andb-sheet 3 of theb-propeller domain in 7 of the16 different integrinasubunits characterized in mammals. TheI domain can be expressed as an isolated domain (12). Thethree-dimensional structure of the I domain shows that it hasa nucleotide-binding fold shared with G proteins and a metalion-dependent adhesion site (13, 14).Several subregions are apparent in integrinbsubunits. Aregion of '240-aa residues (residues 102–344 inb2) shows thehighest conservation between species and among differentsubunits and has been termed the conserved region or con-served domain, although there is little evidence that it is astructural unit. This region has a metal ion-dependent adhe-sion site-like site and has been predicted to fold into an Idomain-like structure (13, 15). However, the exact boundariesand fold of this region are debatable. A fold identical to an Idomain has been proposed for residues inb3that correspondto residues 102–284 inb2(15); however, this omits the last 60residues of the region defined by sequence conservation. Wehave proposed a modified fold that encompasses the entireconserved region (C.H. and T.A.S., unpublished data). Evi-dence that the domain boundaries indeed correspond to theconserved region comes from three mAbs that bind combi-natorial epitopes involving both Arg-133 and His-332, and onemAb that binds both Arg-133 and Asn-339. These residues arein the first and last predicteda-helices in the conserved region,which are predicted to be adjacent in the structure. It is notablethat in the I domain, the first and lasta-helices are alsoadjacent (13, 14). The vast majority of mutations in leukocyteadhesion deficiency I map to the conserved region (see ref. 16).Because these mutations block association ofaandbsubunitsduring biosynthesis of integrins, thebsubunit conserveddomain is important for association witha(8). Site-directedmutagenesis and cross-linking experiments suggest that theconserved region is important in ligand binding (17). Further-more, both function-blocking and activating mAbs map to thisregion (18, 19) (C.H. and T.A.S., unpublished data).A cysteine-rich region is present in the C-terminal portionof thebsubunit extracellular domain, from residues 423 to 609.The 20% content of cysteine suggests that this region is highlyrigid. Interestingly, a number of mAbs that activate or reportconformational changes map to this region inb1,b2, andb3(20–26) (C.H. and T.A.S., unpublished data).Here, we study the folding during biosynthesis of the integrinb2subunit. Previous studies on leukocyte integrin biosynthesishave shown that theaandbsubunit precursors are initiallyunassociated and have high mannose N-linked carbohydrate,and that processing to complex carbohydrate with an accom-panying increase in Mrdoes not occur until afteraandbsubunit association (4, 8, 27, 28). Thus, transport from theendoplasmic reticulum to the Golgi apparatus is dependent onformation of theabcomplex. In general, the leukocyteintegrinbsubunit is produced in excess over theasubunits andrequires substantially longer to be chased from the precursorto the mature form. We demonstrate by reactivity with mAbThe publication costs of this article were defrayed in part by page


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