2. Membrane-spanning proteins are precisely localized at sites of physiological function: what is the code? NaV CNG- CHANNEL INITIAL SEGMEN T NODE OUTER SEGMENT Na/K ATPase E-CADHERIN INITIALSEGMENT NODE DYSTROGLYCAN ATAXIA ARRHYTHMIA MUSCULAR DYSTROPHY BLINDNESS
4. Channels/transporters Anion exchanger Na/K ATPase Volt. Sens. Na channel Na/Ca exchanger IP3 receptor RhBG NH3 transporter KCNQ2/3 Kv3.1b CNG-beta Kir6.2 (K (ATP) channel) Cell adhesion molecules CD44 L1 CAMs E, N-cadherins dystroglycan Ankyrins-G, -B and-R and their partners Recognition by ANK repeats developed independently multiple times in evolution ANK repeats
5. 480/270 kDa Ankyrin-G co-clusters with volt.-gated Na channels and 186 kDa neurofascin at axon initial segments and nodes of Ranvier JCB 110:1341-52 (1990) JCB 114:1243-59 (1991) JBC 270:2352-59 (1995) JCB 135:1355-67 (1996) JNeurosci.17:7025-36 (1997) Steve Lambert Katya Kordeli
6. Cerebellar knockout of ankyrin-G: severe ataxia and loss Of ability of Purkinje neurons to fire action potentials Zhou et al., J. Cell Biol. 143: 1295-1304 ( 1998)
7. Ankyrin-G (-/-) Purkinje neuron initial segments: coordinate loss of Nav1.6, beta-4 spectrin, and neurofascin +/+ -/- -/- Jenkins and Bennett, JCB 155: 739-46 (2001)
8. Josh Huang: Ango et al. Cell 119:257 (2004) An ankyrin-G-based Gradient of Neurofascin directs Interneuron synapses At Purkinje neuron Axon initial segments
9. KCNQ 2/3 require ankyrin-G to co-localize with Na channels at axon initial segments Ed Cooper: Pan et al. (2006) J. Neurosci.26:2599 Wildtype AnkG null
10. Christian Shultz: Sobotznik et al. PNAS 106:17564 (2009) Ankyrin-G-null axon initial segments become dendrites
11. Loss of fasciculated microtubules in ankG-null AIS: cytoplasmic role for 480/270 kDa ankG inserted residues
12. ankyrin-G/beta-4 spectrin as a common element in establishing axon initial segment synapses with interneurons Howard et al., (2005) Trends Neuroscience 28:310
13. Chandelier interneuron synapses at axon initial Segments: final stage of development Howard et al., (2005) Trends Neuroscience 28:310
14. Selective alterations in postsynaptic markers of chandelier cell inputs to cortical pyramidal neurons in subjects with schizophrenia. Cruz et al. (2009) Neuropsychopharmacology 34:2112-24. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Alarcón et al. (2008) Am J Hum Genet. 82:150-9. Two variants in Ankyrin 3 (ANK3) are independent genetic risk factors for bipolar disorder. Schulze et al. (2008) Mol Psychiatry 14:487-91. Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder.Ferreira et al. (2008) Nat Genet. 40:1056-8. Axon initial segments: Center stage as substrates for Psychiatric disease
15. L1 CAMs: -QFNEDGSFIGQY- - nematode/fly NaV: -VPIAVGESDFE- fruit fly zebrafish KCNQ2: -PYIAEGESDTDSD- dog fish zebrafish KCNQ3: -RYLAEGETDTDTD- dog fish zebrafish ? How difficult is it to evolve ankyrin-binding activity Convergent evolution of ankyrin-binding motifs in components of axon initial segments Family motif absent present
16. Ankyrin-G and Nav1.5 in cardiomyocytes: co-localize at intercalated discs and T-tubules and co-immunoprecipitate Mohler et al. PNAS 101:17533-8 (2004)
17. E1053K mutation of Nav1.5: cardiac arrhythmia (Brugada syndrome); loss of both ankyrin-binding and delivery of Nav1.5 to the cardiomyocyte cell surface Mohler et al., PNAS 101:17533-8 (2004) Imperm. Perm
18. Ankyrin-G co-localizes with E-cadherin in early mouse embryos and is required for compaction Kizhatil et al. J. Biol. Chem. 282:26552 (2007)
19. Ankyrin-G binds to the cytoplasmic domain of E-cadherin and Is required for E-cadherin to accumulate at the cell surface E-cadherin localizes in An intracellular compartment In ankyrin-G-depleted Bronchial epithelial cells
20. Knockdown of ankyrin-G blocks biogenesis of the lateral membrane in telophase (same result with beta2-spectrin)
21. (Ervasti JM. JBC 268(16); 2003) The dystrophin-glycoprotein complex transmits contractile force to the extracellular matrix at costameres DGC is missing from the Sarcolemma in Duchenne muscular dystrophy
22. Ankyrin-B knockout mice: Congenital myopathy and loss of sarcolemmal dystrophin and dystroglycan Tuvia et al. (1999) J. Cell Biol. 147:995 Ayalon et al. (2008) Cell 135:1189
23. Knockdown of ankyrin-B: Intracellular retention of beta-dystroglycan in the juxta-TGN Adult muscle ankB siRNA
24. Control ankG siRNA Ankyrin-G is required to retain the DGC at costameres Gai Ayalon
25. Ankyrins and the DGC: Dp71 dystrophin binds to ankyrin-B and Ankyrin-G; beta-dystroglycan cyt. domain binds to ankyrin-G Dp71 forms a Ternary complex With ankyrins and Beta-dystroglycan
26. Mutation of DP71 causing Becker’s muscular dystrophy reduces binding to ankyrin and localization at costameres
27. Ankyrin-G and dystrophin Dp71 bind to distinct sites on dystroglycan and can form a ternary complex
28. A-B A-G Dystrophin (Dys) Beta-Dystroglycan (DG) ?Transport from TGN to costameres TGN A-B DG Dys
29. Phototransduction requires segregation of the cyclic nucleotide-gated channel to outer segments of photoreceptors Burns and Arshavsky, Neuron 48:387 (2005) spectrin in the inner segment: Madreperla et al., JCB 1989 ? Are ankyrins involved in photoreceptor polarity CNG1
30. Ankyrin-G segregates to the outer segments of rod photoreceptors and ankyrin-B to the inner segments Krish Kizhatil: Science 323:1614 (2009)
31. shRNA-knockdown of ankyrin-G in the neonatal mouse retina eliminates CNG channel subunits in rod outer segments but not rhodopsin Injected-P0; Sacrificed-P 21
32. CNGB1 channel has a C-terminal ankyrin-G-binding site that is eliminated by a retinitus pigmentosa mutation Co-IP from bovine photoreceptor outer segment extracts 1= full length Nf 2= Nf-CNG N-term 3= Nf-CNG C-term 4= Nf-CNG C-term RP
33. Ankyrin-G-binding is both necessary and sufficient to Target human CNG-beta1 to rod outer segments in transgenic tadples
34. Independently evolved ankyrin-binding sites share an unstructured conformation and include tyrosine and dileucine “motifs” RhBG KLP FLD SPP AE1 PAVLTRSGDPS KCNQ2 PYIAEGESDTDSD NaV VPIAG E SDFE Kir6.2 VPIVAE E D E-cadherin KEPLLPPEDDTRDNVYYYDEEGGGEED NF/L1CAMs QFNEDGSFIGQ Y Dystroglycan GVP IIF ADELDDSK CNG beta PEPGEQ IL SVKMPE Hypothesis: ankyrin-G is a general adaptor that reads diverse natively unstructured motifs in polarized cells ANK repeat solenoid: ? Extended groove binds peptides
35. A conserved ankyrin-based mechanism for formation and maintenance of diverse specialized membrane domains that have recently evolved in vertebrates: -Ankyrins function as adaptors for functionally related membrane proteins through easily accessed recognition code Future Questions: What specifies ankyrin localization? What are the effectors that translate ankyrin-binding into cellular localization and domain assembly? Role of ankyrins in cellular organization of signaling pathways Ex TGF-beta and EGF receptors
36. Knockdown of ankyrin-B eliminates a subset of Microtubules associated with costameres Control -Ank-B Green= MT Red= Ank-B
37. Dyn4 Ankyrin-B binds to dyn4/p62 of the dynactin complex and is required for association of dyn4/p62 with costameres: Possible mechanism for costamere localization of microtubules
38. Ankyrin-B-binding activity of Dynactin-4 is required for costamere localization of microtubules and the DGC Dynactin-4/HA -Tubulin Dyn4 siRNA+ HA-Dyn4 Dyn4 siRNA Dyn4 siRNA+ HA-Dyn4 N331A -DG Dystrophin Ankyrin-B Dyn-4 N331A= loss of ankyrin-B binding mutation
