A major effort in our laboratory is to understand the structural basis of integrin activation and signaling using biochemical, structural and animal models. A potential outcome is the discovery of small molecule antagonists to treat common diseases linked to integrin dysfunction.
Crystal structure of the A-type domain from integrin CD11b. Cell 1993;72:287, Cell, 1995;80:631
A ribbon drawing of the crystallized extracellular segment of alpha-V beta-3. The alpha-V and beta-3 subunits appear in blue and yellow, respectively. The structure is severely bent at the kneelike "genu" (arrows). Disordered regions are in gray. At right is a computer model of the straightened alpha-V beta-3, which was developed by extension (135 degrees) and rotation (120 degrees) of the bent structure at the genu. The approximate location and shape of three small and disordered domains are shown in gray. Science, 294:339, 2001
Surface representation of the ligand-binding site in an integrin heterodimer (a subunit is in blue; b subunit is in red) . The ligand is an Arg-Gly-Asp conatining peptide, shown as ball-and stick model. Two metal ions are shown in cyan and magenta. Science, 296:151, 2002
Ribbon diagram of a hypothetical model showing an inactive (left) and an active (right) conformations of the aA-integrin CD11b/CD18. Current Biology, 12:R340, 2002
Pseudoatomic models of unliganded (A) and fibronectin-bound (B) integrin aVb3. aV is in blue and b3 in red. Fn-9 and 10 are in green and yellow, respectively. J Cell Biol 168:1109, 2005
(a, b) Loss of the transcription factor ZBP-89 results in a bloodless phenotype in zebrafish. (a, b) DAF staining of 48 hpf whole-mount zebrafish embryos. Blood (arrows) is present in axial vessels and heart (short arrow) in wild-type (a) but not ZBP-89 morphant (b) embryos. Views are lateral with anterior to the left and dorsal to the top. (c, d) Overexpression of ZBP-89 impairs angiogenesis. In situ hybridization of wild-type embryos (c) or wild-type embryos overexpressing ZBP-89 (d) reveals a marked reduction in intersomitic expression of the endothelial markers flk1 (d), when compared with the respective wild-type embryos. Development, 133:364, 2006
Ribbon diagram showing two views of a structure model of the complete aVb3 ectodomain plus the TM domains. The orientation of the ectodomain relative to the TM domains, show that the ligand binding site is accessible to macromolecualr ligands without unbending. The a-genu and propeller metal ions are in orange. J Cell Biol, 186: 589, 2009
M. Amin Arnaout, MD
Professor of Medicine, Harvard Medical School
149 13th Street, Charlestown, MA 02129
A. Cell-matrix interactions: Structure-activity relationships in integrins.
Cells exist in a highly dynamic extracellular milieu consisting of complex chemicals and mechanical stress signals to which cells must continuously adjust and in turn moderate. In metazoa, the task of integrating these mechanochemical cues across the plasma membrane is divalent-cation-dependent and is mediated by integrins, αβ heterodimeric type I membrane receptors. Integrins are normally expressed in a low affinity state, but rapidly and reversibly switch into high affinity by agonists, which act on the integrin cytoplasmic tails to modify their affinity to extracellular ligands (so-called inside-out activation). Ligands bind activate integrins eliciting classic "outside-in" signals that regulate every aspect of cell function. We have identified a human disease, leukocyte adhesion deficiency, in which a sub-group of integrins, b2 integrins, is lacking. The affected patients suffer from life-threatening bacterial infections. Loss of other integrins causes bleeding diathesis or organ malformations. Improper activation of integrins on the other hand is associated with common inflammatory and metabolic disorders including atherosclerosis, diabetes, and cancer metastasis. A major effort in our laboratory is to understand the structural basis of integrin activation and signaling using biochemical, structural and animal models. A potential outcome is the discovery of small molecule antagonists to treat common diseases linked to integrin dysfunction.
B. Autosomal Dominant Polycystic Kidney Disease
ADPKD is the most common monogenic disease in humans, caused by dysregulation in diameter of tubular structure including kidney tubules and blood vessels leading chronically to loss of renal function or cutely to cerebral hemorrhage due to ruptured vascular cysts. We have generated a mouse KO of ADPKD and demonstrated that one of the two defective genes, PKD2, encodes a TRP-like calcium channel, which is stabilized by the product of the second gene, PKD1. We have identified a transcriptional modulator of PKD1, which causes pronephric cysts when knocked down in zebrafish. Biochemical and cell biology approaches are being used to elucidate the pathway linking this modulator to cystogenesis in zebrafish and mouse models. Other work attempts to elucidate the signaling pathways involved in determining tube diameter.
C. Developmental Biology - fate determination of hemangioblasts
This is a new area of investigation in my laboratory, driven initially by serendipitous findings I made while pursuing aspects related to integrins. We found that a zinc finger transcription factor, which regulates a b2 integrin in mature leukocytes, appears to regulate developmental fate of hemangioblasts, the precursors of hematopoietic and vascular stem cells. Work is ongoing to define how each lineage is regulated by this factor at the transcriptional level and the interacting proteins involved using embryonic stem cell/embryoid body cultures, siRNA, and zebrafish and conditional mouse KO models.
Michishita M, Videm V, Arnaout MA. 1993. A novel divalent cation-binding site in the A- domain of the beta 2 integrin CR3 (CD11b/CD18) is essential for ligand binding. Cell. 72: 857.
Lee JO, Rieu P, Arnaout MA (Corresp. author), Liddington R. 1995. Crystal structure of the A domain of the alpha subunit of integrin CR3 (CD11b/CD18). Cell. 80:631.
Kim K, Drummond I, Ibraghimov-Beskrovnaya O, Klinger K, Arnaout MA. 2000. Polycystin 1 is required for the structural integrity of blood vessels. Proc. Natl. Acad. Sci. USA. 97; 1731.
Gonzalez-Perrett S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M, Harris PC, Reisin IL, Arnaout MA (Corresp. Author) and Cantiello HF. 2000. Polycystin-2, the protein mutated in autosomal dominant Polycystic Kidney Disease (ADPKD), is a Ca2+-permeable non-selective cation channel. Proc. Natl Acad. Sci. USA. 98; 1182.
Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, and Arnaout MA. 2001. Crystal structure of the extracellular segment of integrin alphaVbeta3. Science. Oct 12; 294(5541):339.
Xiong JP, Stehle T, Zhang R, Dunker R, Joachimiak A, Frech M, Goodman SL, and Arnaout MA. 2002. Crystal structure of the extracellular domain of integrin alphaVbeta3 in complex with an Arg-Gly-Asp Ligand. Science. Apr 5; 296(5565): 151-155, 2002.
Alonso J, Essafi M, Xiong JP, Stehle T, and Arnaout MA. 2002. Does the integrin alphaA domain act as a ligand for its betaA domain? Current Biology. 12: R340.
Adair BD, Xiong JP, Maddock C, Goodman SL, Arnaout MA (co-Corresp. Author), Yeager M. 2005. Three dimensional EM structure of the ectodomain of integrin alphaVbeta3 in a complex with fibronectin. J. Cell Biol. 168(7): 1109.
Li X, Xiong JW, Shelley CS, Park H, Arnaout MA. 2006. The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells. Development. 133(18): 3641.
Xiong JP, Mahalingham B, Alonso JL, Borelli LA, Riu X, Zhang, Anand S, Hyman BT, Rysiok T, Muller-Pompalla D, Goodman SL, and Arnaout MA. 2009. Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment. J Cell Biol. 186(4): 589.
Arnaout, MA. 2010. Autosomal Dominant Polycystic Kidney Disease. In Encyclopedia of the Life Sciences. 2010, John Wiley & Sons, Ltd, UK. DOI: 10.1002/9780470015902.a0006010.pub2
12. Mahalingam B, Ajroud K, Alonso JL, Anand S, Adair B, Horenstein AL, Malavasi F, Xiong, JP, and Arnaout, MA. Stable coordination of the inhibitory Ca2+ ion at the metal-ion-dependent-adhesion site in integrin CD11b/CD18 by antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design. J. Immunol. 2011. 187; 6393.