lecture 4- laminin and integrins

laminin is major component of BM that self assembles into network to present binding sites for cells

• laminin is high molecular weight glycoprotein(800kDa)

• after collagen IV, laminin is most abundant BM protein

• originally isolated from tumour in rodents. made of basement membrane components

• crucible structure

laminin made of 3 chains into crucible structure

• 3 separate polypeptide chains

• alpha c terminal makes globular domain

• coiled coil, different from collagen

• each chain made of alpha helix

• alpha helices wrap together to form long arm

laminin 1 spontaneously forms a network in vitro

• self assemble into network of laminin molecules interacting with other laminins

alpha chain golbular domain contains 5 LG domains that interact with cell surface receptors

• 5 subdomains which are binding sites for different cell surface receptors

• 3D structure assemble into network, cell binding sites stick out

laminin network is linked with collagen IV network by accessory molecules

• laminin form binding sites

• collagen is foundation

many laminin genes but only limited number of trimers are formed

• 11 laminin genes form 15 different heterotrimeric combinations

• more laminin isoforms than collagen IV isoforms

• more variability

different laminin isoforms show tissue specific expression

• some are widely expressed, in early embryos

• mice unable to develop where basement membrane forms

• other laminin genes show tissue specific defects when they are deleted in mice, phenocopy some human diseases

loss of laminin beta 2 isoform leads to similar condition as loss of GBM collagen IV isoforms

• pierson syndrome- rare lethal condition

• congenital nephrotic syndrome progressing to end stage renal disease

• eye abnormalities

• sever muscular hypotonia

• laminin 11(alpha5beta2gamma1) expressed in GBM, eye and synaptic BM, explains disease phenotype

• frame shift mutation

• glomeruli don’t filter properly, similar to alports

• affecting tissue where laminin trimer has key role

mice deficient for laminin beta 2 gene phenocopy pierson syndrome

• beta 2 deficiency GBM shows beta 1 chain expression

• GBM contains wrong laminin isoform, can’t form effective filtration barrier

• collagen IV not affected

• laminin in glomerular BM doesn’t provide filtration barrier

• need to be in right isoform to have functional filtration

specific laminin isoforms in epidermis link to underying collagen in dermis

• epidermis is attached to underlying dermis via basement membrane

• mechanically strong

• epidemolysis bullosa- group of conditions where skin blisters following mechanical trauma, mutations affect mechanical strength of dermal/epidermal junction, position of the break depends on genetic defect

• 3.5kg of skin held on by laminin sticking to integrin

• severity of blistering depends on where it happens

laminin 5(alpha3, betas3, gamma 2) in skin BM

• links cell surface adhesion proteins in structures called hemidesmosomes to underlying collagen

• collagen VII are anchoring fibres that link BM to the collagen I network in dermis

• collagen VII is specific to basement membranes

• collagen VII is out of BM, links to collagen I fibres in dermis

• hemidesmosomes- dense patches, cell surface receptors are clustered, attach to cytoskeleton

laminin 5(a3, b3,y2) mutations cause junctional EB

• complete loss of any laminin V chain leads to herlitz type JEB, lethal within the first few months after birth

• other mutations with pertubed laminin 5 function leadws to milder forms of condition

• autosomal recessive conditions

• no laminin in epidermis, skin has no mechanical strength, lifts off dermis

• non-herlitz is partial loss of function mutation, can survive but severe blistering

genetic models in mice prove that loss of laminin 5 causes junctional EB

• laminin 5 deletion in mice phenocopies herlitz JEB

  • no difference at birth

  • develop blisters post natal

  • die by day 3

• knockout one critical gene for mice

• shortly after birth, mouse dies, dehydrates

• important to have mechanical connection

gene therapy for laminin beta 3 chain mutations

• 7 year old with splice site mutation in exon 14 of LAMB3

• suffered severe blisters since birth, presented at hospital following S aureas infection, loss of 60% of epidermis

• used retrovirus to deliver functional LAMB3 gene to patients own keratinocytes

• holoclones are proliferative and contain stem cells

• after 8 months skin was almost entirely derived from holoclones

• skin repopulated by holoclones(stem cell regions)

• replaced defective laminin with functional gene

cell surface receptors for laminin link epidermal cells to BM

• cell-ECM junctions in skin

  • structural links between cytoskeleton and matrix

  • provides physical strength to tissues

• continual linkage

• everything in cell mechanically connected

integrins

• they are cell/ECM adhesion receptors on most cells

• heterodimers of alpha and beta subunits

• large extracellular domain

• single transmembrane spanning domain

• short cytoplasmic domain( exception in beta 4)

• 2 legs go to plasma membrane

• alpha and beta subunits specifies what integrin it will bind to

• very short tail that will connect to cytoskeleton

alpha and beta subunits

• numerous alphabeta heterodimers

• distinct and overlapping specificity for different ECM

• RGD(Arginylglycylaspartic acid) receptors interacts with ECM glycoproteins

integrin cytoplasmic domain and cytoskeleton

• most integrins associate with actin

• in hemidesmosomes, link to intermediate filaments made of keratin

• keratin is imporant for epidermolysis bullosa

• connect ECM with cytoskeleton

• integrin binds to actin

• keratin fibres used to resist mechanical force

tissue specific distribution of beta integrin isoforms

• integrin is tissue specific

• beta 1 expressed in early embryos

hemidesmosome specific beta 4 integrins are required for integrity of the skin

• deletion of beta 4 integrins leads to same phenotype, like laminin 5 deletions

loss of beta 4 integrin in mice

• leads to loss of hesmidesmosomes, but not cell/cell adhesions that link to IF

• not attaching to basement membrane, whole sheet comes off

junctional epidermolysis bullosa with pyloric atresia (PA-JEB)

• rare autosomal recessive condition associated with loss of a6b4 integrin

• neonatal mucocutaneous blistering and gastric outlet obstruction through loss of function in GI, genitourinary and respiratory epithelium

• can also lead to atresia- problem with digestive tract

• disease is fatal

• PA-JEB patient with 2 distinct mutations in beta 4 integrin alleles

  • paternal mutation leads to shift in open reading frame, downstream premature termination codon

  • maternal mutation leads to donor splice site, in-frame exon skipping

loss of beta 4 can lead to absence of alpha 6 integrin subunit

• loss of alpha 6 integrin expression

• formation of basement membrane still occurs, presence of laminin 5

• able to make basement membrane, but unable to attach

tissue specific distribution of alpha integrin isoforms

deletion of alpha 6 integrin has same affect as loss of beta 4 in mice and humans

• loss of chain, same as loss of lamina

EB simplex has milder phenotype than other forms of EB

• usually restricted to blisters on regions subject to mechanical stress

• can deal without significant scarring

• compare with JEB, has 40% mortality in first year

• mostly associated with mutations in keratin 5 and 14

deletion of beta 4 cytoplasmic tail in mice results in EB simplex

• cells are weakened internally, but BM is intact

• cells rip apart leaving part still attached to BM via integrin

• 49 year old with heterozygous 2bp deletion in ITGB4 leads to skipping exon, leads to 50 amino acid deletion in cytoplasmic domain, integrin binds to IF cytoskeleton