ADAM gene expression in the adult CNS and genetic aberrations in cancer cells Ari Huovila Institute of Medical Technology University of Tampere and Tampere University Hospital, Tampere, Finland [email protected] www.uta.fi/imt Abstract ADAM metalloprotease-disintegrins share a common modular structure of functional domains for proteolytic, cell adhesion, and signaling interactions. The metalloprotease domain of roughly half of the known ADAMs contain an intact consensus metzincin catalytic site, and they are thus thought to function as active metalloproteases. The types of interactions mediated by ADAMs are expressly conspicuous in the CNS. However, the information of ADAM functions in the adult CNS remains fragmentary while the neural ADAM research has mainly been focused at the neural development. Nevertheless, ADAMs are emerging as a major CNS metalloprotease family, implicated in, e. g., alpha-secretase processing of amyloid precursor protein (APP), proteolytic activation of cytokines and growth factors, and ectodomain shedding of cell surface receptors. An important prerequisite for exploration of individual ADAMs is detailed knowledge of their expression in the CNS. Here, an update on CNS ADAM gene expression will be presented, including a novel potentionally proteolytic ADAM. Their dual potential to mediate both proteolysis and cell-cell contacts raises also the question about the involvement of ADAMs in the pathobiology of malignancy. Indeed, ADAMs have been associated with several types of cancer. Here, novel data on genomic rearrangements and aberrant pre-mRNA splicing of ADAM15 will be presented, suggesting that one of the impairments in the genetic machinery in cancer cells may be at the level of post-transcriptional processing. ADAM proteins (a disintegrin and metalloprotease) - also known as MDCs (metalloprotease-like, disintegrin-like, cysteine-rich) - modular domain structure - type I transmembrane proteins (except some alternatively spliced isoforms) - metalloprotease domain: about half of the ADAMs contain an intact metzincin-type active site ⁄ about half of the ADAMs are not active proteases - adhesion domain: all ADAMs contain a putative integrin-binding (disintegrin) motif - signaling motifs in the cytosolic tails - over 30 ADAM genes found, from C. elegans to man - widespread expression, in all tissues and cell types in mammals - over 20 related ADAM-TS genes ADAM PRECURSOR SS PRO MP DI CYS EGF TM CT ADAM2 Q L L S L S M G T T Y D ADAM2 C R P S F E E C ADAM9 H E L G H N L G M N H D ADAM9 C R G K T S E C ADAM10 H E V G H N F G S P H D ADAM11 C R E A V N E C ADAM12 H E L G H N F G M N H D ADAM12 C R D S S N S C ADAM15 H E L G H S L G L D H D ADAM15 C R P T R G D C ADAM17 H E L G H N F G A E H D ADAM23 C R D A V N E C ADAM proteins can • Proteolytically process other proteins - biosynthetic processing of nascent proteins - activation/inactivation of growth factor and other effector proteins or receptors (ectodomain shedding) - remodeling other proteins, e. g., ECM • Mediate cell-cell adhesion - by binding to integrins on the adjacent cell membrane via the disintegrin domain - by binding to other counter-receptors/ligands (syndecans) • Relay signals - several ADAM cytosolic domains contain various signaling motifs (e. g., SH2- and SH3-ligands) Part 1: ADAMs in the CNS ADAMs in the CNS - the types of molecular and cellular interactions mediated by ADAMs are particularly manifest in the CNS - at least 17 ADAM genes are expressed in mammalian CNS (also several ADAM-TSs) - ADAMs implicated in both physiological and patholo- gical events in the CNS - neural differentiation, migration, axon guidance, APP and prion processing, cell adhesion, neuro- protection - individual ADAM functions in the adults CNS remain largely to be investigated ADAM genes expressed in the adult CNS* a. k. a. adhesion MP substrates cytosolic inter- actions ADAM1 fertilin-αα9β1 ADAM2 fertilin-βα6β1, α9β1 ADAM3 cyritestin α9β1 ADAM4 ADAM5 ADAM7 ADAM8 CD156 (n. d.) MBP, (IL-1Rc, TNF-α) ADAM8 ADAM9 meltrin-γα6β1, α9β1, hbEGF, (APP), fibro- SH3PX1, MAD2β, αVβ5 nectin, collagen XVII endophilin I ADAM10 MADM, Kuzbanian, ephrin-A2 APP, Notch, Delta, L1, (α-secretase) (ephrin-A2), Slit,TNF-α, collagens IV/XVII, prion ADAM11 MDC, MDC1 ADAM12 meltrin-αα9β1, syndecans IGFBP-3, IGFBP-5 Src, Grb2, α-actinin-1 and -2 ADAM15 metargidin α5β1, α9β1, αVβ3 (in vitro) SH3PX1, MAD2β, Endophilin I, ”Src’s” FAP52/PACSIN2 ADAM17 TACE TNF-α , (APP), IL6R, MAD2MAD2 TGF-α, ErbB4, CD30, L-selectin, TRANCE, p75 TNFR, fractalkine ADAM19 meltrin-β neuregulins ADAM21 ADAM22 brain MDC2 14-3-3zeta ADAM23 brain MDC3 αVβ3 ADAM33 *the molecular interactions listed in the table are mostly demonstrated in non-neural context Potential metalloprotease Inactive metalloprotease domain ADAMs in the adult brain, example 1 APP α-secretase - several proteins are capable of APP α-processing: ADAM9, ADAM10, ADAM17, prohormone convertase 7 - ADAM10 and ADAM17 the prime candidates - accumulating literature corroborates ADAM10’s role as the α-secretase: - brain expression pattern more consistent with ADAM10 as the α-secretase (Kärkkäinen &c. MCN -00; Marckinkiewicz & Seidah JNC -00) - cellular location consistent with ADAM10 as the α-secretase (Marckinkiewicz & Seidah JNC -00; Goddard &c. Glia -01; Hurtado &c. JCBFM -02) - evidence for ADAM17 involvement (Buxbaum &c., JBC -98; Slack &c., Biochem J -01) ADAM10 ADAM17 (TACE) Kärkkäinen &c. MCN 2000 ADAM10 mRNA ADAM17 mRNA Telencephalon olfactory bulb ++ - piriform ctx ++ - frontoparietal ctx ++ - lateral septum + - hippocampus CA1-CA4 ++ + dentate gyrus ++ + Diencephalon zona incerta + - subtalamic nucleus + - hypothalamus ventromedial nucleus + - dorsomedial nucleus + - arcuate nucleus + - Mesencephalon inferior colliculus ++ +++ Cerebellar ctx Pons/medulla +++ ++ pontine nuclei - ++ Kärkkäinen &c. MCN -00 ADAMs in the CNS example 2 ADAM10 in axon guidance - regulated cleavage of a contact-mediated axon repellent ephrin-A2 (Hattori &c. Science -00) - ADAM10 binding to ephrin-A2 - ADAM10 mediated proteolysis of Ephrin-A2? - regulation of midline crossing of CNS axons (Schimmelpfeng &c. Mech Dev -01) - ADAM10 mediated cleavage of Slit/Roundabout complex? EphA3 ADAM10 ephrin-A2 axon glia further potential CNS ADAM functions CNS ADAM1 and ADAM2 - implicated in AD (Gerst &c., J Neurosci Res 2000) ADAM8 (CD156) - DI domain of ADAM8 enhances protection vs. experimental autoimmune encephalomyelitis, neuritis, and uveitis (Scluesener, J Neuroimmunol, 1998) - highly induced in affected areas of Wobbler (WR) mice: cell adhesion during neurodegeneration (Schlomann &c., JNS -00) ADAM10 - Notch/Delta activation (Rooke &c., Science -96; Pan & Rubin, Cell -97: Qi &c. Science -99; ) - prion protein processing (Vincent &c. JBC -01) ADAM15 - neural regeneration (Bosse &c., Glia -00) … further potential CNS ADAM functions CNS ADAM17 -TACE iNOS activation (Hurtado &c., Neuropharmacology -01; Hurtado &c., JCBFM - 02 - Notch-receptor processing (Brou &c., Mol Cell -00) - fractalkine shedding, &c. - prion protein processing (Vincent &c., JBC -01) ADAM19 - neurogenesis? (Kurisaki &c. Mech Dev -98) ADAMs 11, 22, 23 - integrin counter-receptors (Sagane &c., Biochem J, Gene -99; Rybnikova &c., NSC -02) - axon guidance (Leighton &c., Nature -01) ADAM33 expression in mouse CNS • mouse chr2; human chr20p13 (Gunn&c. -02; Yoshinaka&c. 02) • 22 exons, 797 amino acids (mouse) • closest homology to ADAM13 > ADAM12, ADAM19; ectodomain most similar to ADAM12 (Kärkkäinen &c., subm.) • putatively active metalloprotease • expressed in several tissues (RT-PCR; Gunn &c. -02; Yoshinaka &c. -02) • CNS expression (ISH, Kärkkäinen &c., subm.): - no embryonic expression - post-natal development - first detected at P8 - olfactory bulb, neocortex (IV layer) - transiently in cerebellar cortex (P12) - adult: olfactory bulb, neocortex, not in hippocampus - neuronal expression (granular neurons) • an ADAM metalloprotease subfamily (ADAMs 7, 8, 13, 12, 15, 19, 28, 33)? (Kärkkäinen &c., subm.) A D A M A A 1 D D 7 A A M M ADAM A 1 1 D 0 4 A A D M A 1 1 M A 9 2 D 1 3 A A M 1 D 3 1 A 3 M M A 15 D 22 A M AD ADA A M8 23 AM AD ADA M28 32 AM AD ADAM7 ADAM2 ADAM30 ADAM5 ADAM29 A DAM 18 20 AM A AD D A AM 21 D 3 M A A M D 9 A 5 AD 2 M A A A M A D D 1 D A A A AM a A D M A D A 2 D 1 4 M A A a M 2 M 6 A 3 A 1 4 D 6 D A A M M 4 6 ADAM33 expression in the adult mouse CNS A) ADAM33 mRNA in the 4th layer of frontal cortex and in the granular neurons of the olfactory bulb B) ADAM33 mRNA in granular neurons of the frontal cortex (layer 4) The dead metalloproteases: ADAM11, ADAM22, ADAM23 - ADAM11, ADAM22, ADAM23: the brain MDCs (Sagane &c. Biochem J -98, Gene -99) - ”inactivated” catalytic site (Cerretti &c., BBRC -99) - predominantly CNS expressed - integrin counter-receptors in the CNS? - ADAM23 binds αVβ3 (Cal &c., MBC -00) - disintegrin domains/motifs very conserved shared specificity? - ADAM23 implicated in axon guidance (Leighton &c., Nature -01) X predominantly CNS expressed ”brain MDC” genes (ADAMs 11, 22, and 23) group together, separately from other ADAM genes The ”brain MDCs” HADAM 11 C K Y E P R G V S C R E AVN E C D I A E T C HADAM 22 C K F Q PMG T V C R E AVN D C D I R E T C HADAM 23 C LFQ P R G Y E C R D AVN E C D I T E Y C MADAM 11 C K Y E P R G V S C R E AVN E C D I A E T C MADAM 22 C K F Q PMG T V C R E AVN D C D I R E T C MADAM 23 C LFQSR G Y E C R D AVN S C D I T E Y C ADAM11 (MDC1) ADAM22 (MDC2) ADAM23 (MDC3) Brain region mRNA Brain region mRNA Forebrain Thalamus Telencephalon Ante rodorsal