Ankyrins

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Ankyrins

• Foundation Conference I
• January 6, 2003

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Cytoskeleton on the Cytoplasmic Side of the Human
Red Blood Cell Membrane
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Organization of the Erythrocyte Membrane Skeleton
Ankyrin in human erythrocytes provides a
high-affinity link between the cytoplasmic domain
of the anion exchanger and the spectrin/actin
network
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Ankyrin Family in Mammals

• Ankyrins are members of a family of three closely
  ralated genes

• Ankyrin-R (restricted, encoded by ANK1)
  primarily found in erythrocytes, skeletal muscle
• Ankyrin-B (broadly expressed encoded by ANK2)
  first characterized in brain, the major form of
  ankyrin in brain
• Ankyrin-G (giant encoded by ANK3) the most
  widely expressed ankyrin, the major isoform in
  kidney and other epithelial tissues

• Ankyrins are expressed in most tissues, and in
  many cases all three ankyrins are found in the
  same cell type

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Ankyrin isoforms and tissue distribution
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Ankyrin Domains

• 1. Three conserved domains (found in canonical
  ankyrins)
• N-terminal membrane-binding domain (MBD)
  comprised of 24 copies of 33 residue ANK repeat
• Central spectrin-binding domain, 62 kDa
• Death domain 12 kDa, no known role within
  ankyrins
• 2. C-terminal domain divergent, regulatory
  function
• 3. Specialized domains (found in giant ankyrins
  targeting axons)
• Tail domain inserted between spectrin binding
  domain and death domain providing an extended
  random coil up to 0.5 mm in length
• Ser/Thr rich domain

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Ankyrin genes are expressed and processed in a
complex tissue-dependent and developmentally
regulated fashion
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Ankyrin Repeats

• ANK repeats are 33-residue motifs involved in
  protein recognition and are found in more than
  300 unrelated human proteins
• ANK repeats fold into stacks of antiparallel
  a-helices interconnected by exposed loops
  arranged perpendicular to the a-helices.

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• The specificity for ANK-repeatprotein
  interactions is likely to be conferred by
  non-conserved residues that flank each ankyrin
  repeat, located at the tips of exposed loops.

• Ankyrin MBDs with 24 ANK repeats are multivalent
  and can accommodate multiple protein
  interactions thus, they may assemble
  multiprotein complexes at specific cellular sites.

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Ankyrin-associated Proteins
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Ankyrin Functions
The ankyrin family has a general role as an
adapter or a linker between a variety of integral
membrane proteins and the spectrin-based skeleton
and thereby involves in targeting diverse
proteins to specialized membrane domains in both
plasma membrane and the endoplasmic reticulum
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Hereditary Spherocytosis (HS)
Normal
HS

• HS is the most common inherited haemolytic
  anaemia in Northern Europeans
• Caused by primary defects in proteins that link
  the membrane skeleton to the overlying lipid
  bylayer in the RBC cell membrane
• Ankyrin-1 (AnkR) mutations are a major cause (35
  to 60) of dominant and recessive HS (Eber et al,
  1996)

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Model of Ankyrin-R Mutation in Mice

• The nb/nb mutation (chromosome 8) in mice induces
  a near loss of ankyrin-R (210 kDa) in multiple
  cell types including erythroid and neural tissues
• The nb/nb phenotype (White et al, 1990 Peters et
  al, 1991)
• - Severe hemolytic anemia similar to human HS
  with ank-R defect
• - Degeneration of a set of Purkinje neurons
• - Cerebellar dysfunction

• The mouse and human diseases result from
  mutations in homologous loci
• Ank-1 is also specifically required for Purkinje
  cell stability

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Functions of Ankyrins in Non-erythroid Cells

• Role in assembly of domains rich in Na channels
  AnkG clusters voltage-gated Na channels at
  neuromuscular junctions and at axon initial
  segments
• Role of AnkB in targeting of Ca2-release
  channels to the Ca2 compartment of the ER
• Development and morphogenesis
• Control cell proliferation ?
• Role in intracellular sorting and trafficking

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Ankyrin-G is Required for Clustering of
Voltage-gated Na Channels at Axon Initial
Segments and for Normal Action Potential Firing
-/-
/
(Malhotra et al, 2000)
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Ankyrin-B is Required for Proper Intracellular
Targeting of Calcium Homeostasis Proteins
Ankyrin-B-deficient mice (Tuvia et al, 1999)

• Musculoskeletal abnormalities disorganized
  sarcomeres, elevated creatinine kinase levels
• Highly irregular cytosolic Ca waves and reduced
  Ca uptake in cardiomyocytes
• Abnormal localization of SR/ER Ca ATPase (SERCA)
  and ryanodine receptors in cardiocytes, skeletal
  muscle, and immune system

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Ankyrins and Development and Morphogenesis

• AnkB gene K/O results in abnormal development and
  lethal postnatal phenotype
• AnkB (-/-) mice exhibit brain malformations
  resembling defects caused by L1-CAM mutations

(Scotland et al, 1998)
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Other Functions of Ankyrins in Intracellular
Sorting and Trafficking

• Spectrin-ankyrin complex bridges cargo vesicles
  and the dynein-dynactin complex along
  microtubules
• A specific Ankyrin-G isoform is required for
  transport of Na/K-ATPase from the ER to Golgi
• Interacts with other intracellular vesicles
  Ankyrin-G without NH2-terminal repeats found in
  lysosomes of mouse macrophage

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Perspectives

• Functions of ankyrin domains death domain,
  regulatory domain (C-terminal domain)
• Functions of ankyrins outside erythrocytes
• Expression and function of differentially spliced
  isoforms, e.g. truncated forms found in
  intracellular organelles
• The newly recognized role of ankyrin-B in
  targeting proteins to the Ca2 compartment
  suggest potential role for modulating functions
  of immune system, the heart, and nervous system