L4.Cell-Matrix Interaction

Question 1

What characterizes multi-adhesive proteins (also known as cell adhesion proteins)?

Answer 1

1. ECM proteins that have multiple binding domains

2. Function as bridges between cells and other ECM components

Question 2

• What 2 forms does the multi-adhesive protein fibronectin (FN) take?
• How is each form made and where are they found?

Answer 2

1. Insoluble Fibers: Formed by fibroblasts. Found in loose CT, areas of cell migration/development, and areas of wound healing.
2. Soluble Form: Formed by liver. Found in high rates in blood plasma where it binds to fibrin in blood clotting. Found in other fluids

Question 3

What is the very basic Fibronectin Structure?

Answer 3

ECM protein DIMER connected by disulfide bonds at C-terminal. Has specific binding properties located in Domains and sub-domain modules.

Question 4

How many possible FN variants are there due to alternative splicing? What are the two sites of alternative splicing seen (3 exons)?

Answer 4

There are 20 possible cariants for EACH Subunit and therefore 400 possible variants for Dimer.

1. Exons IIIB and IIIA can be included or not. Package deal = 2 possible variants.
2. Exon IIICS (also called Exon V for Variant exon) can be spliced 5 ways!

Question 5

What do FN Type I and II modules each bind to?

Answer 5

Type I module = Fibrin and Heparin

Type II module = Collagen Module; binds to collagen and gelatin (solubilized collagen)

Question 6

What does FN Type III module bind to?
-Explain both binding sub-modules and why each structure permits the binding it does.

Answer 6

1. FN-Cell (Integrin) Binding domain = Type III modules 9 & 10. Each = beta-sandwich structure
   - Module 10: has the Arg-Gly-Asp (RGD) aa sequence that binds to specific integrin on cell surface.
   - Module 9: Synergistic sequence
2. Heparin/HS-PG binding domain = Type III modules 13 & 14. Both are characterized by BASIC aa (Arg & Lys) on one side, this provides a POSITIVELY charged surface for interacting with the NEGATIVELY charged Heparin Sulfate Chains (Neg b/c of Phosphates and carbonates).

Question 7

Osteopontin is a cellular binding protein. 1/3 of its aa are acidic (Asp/Glu) –> Negative charge. Osteopontin is highly glycosylated and phosphorylated. Osteopontin has 3 binding domains. What cellular process is osteopontin involved in? How do two two of its binding domains help with this?

Answer 7

Osteopontin is involved in bone resorption.
-Calcium binding domain: Allows osteopontin to bind to mineralized bone matrix.
-RGD-containing domain allows binding to several different integrens.
These two binding domains give osteopontin the ability to bind cells to bones.

Question 8

Where is Laminin found? Briefly describe Laminin’s structure (including 2 important binding modules) and how it helps laminin perform its function.

Answer 8

Laminin is found in basement membranes. It is the primary cell adhesion proteins in this region.

• Laminin is a crossed shaped TRIMER similar to a lower case t. It has alpha, beta, and gamma chains that vary b/t members of the laminin proteins.
  1. beta chain has Collagen Type IV binding module
  2. Integrin Binding Domain does NOT contain RGD aa sequence.

Question 9

What are the 4 main protein components of a basement membrane? Think about interaction diagram from class.

Answer 9

1. Type IV collagen (forms lattice framework)
2. Laminin
3. Perlecan (HS-PG)
4. Entectin (Sulfated Glycoprotein)

Question 10

What are two differences between GF receptors and Integrins?

Answer 10

1. Integrins have a lower binding affinity b/c there are so many of them that the aggregate binding affinity is high enough and don’t want cells attached to ECM forever.
2. Integrins DO NOT have innate KINASE ability like GF receptors (such as receptor tyrosine kinase) do. Must utilize intracellular kinases such as Focal Adhesion Kinase.

Question 11

What are 3 important alpha-beta integrin heterodimers that bind to fibronectin, laminin, and osteopontin?

Answer 11

1. a5-B1 = Fibronectin
2. a6-B4 = Laminin
3. aV-B3 = Osteopontin - plays role in bone resorption and osteoclast binding

Question 12

Why are Focal Adhesions important? What 3 components make them up?

Answer 12

Focal adhesions are signalling hubs that concentrate and direct signalling proteins.
-Fibronectin-Integrin-Actin microfilaments (in fibroblasts) form the core of focal adhesions

Question 13

Vascular Endothelial Growth Factor (VEGF) has been shown to be ineffective when no ECM is present. Why might this be? What further implications might this have?

Answer 13

VEGF must be attached to a proper ECM (laminin) before it can cause endothelial cells to differentiate into capillaries. May have implications for tumor treatments.

Question 14

What cell adhesive protein and integrin receptors are necessary for assisting osteoclasts in bone resorption?

Answer 14

Osteopontin is necessary b/c it attaches to the hydroxyapatite via its calcium binding domain and to the aV-B3 integrin on osteoclasts. This puts the osteoclast in contact with bone to allow it to carry out bone resorption.

Question 15

What are the 6 components of the Hemidesmosome cell adhesion structure?

Answer 15

1. Keratin Int Filament
2. Adaptor Plaque Proteins
3. a6-B4 integrin
4. Laminin 5
5. Basement Membrane
6. Collagen VII

Question 16

What is epidermolysis Bullosa? What gene mutation would cause the most severe form of Epidermolysis Bullosa?

Answer 16

Epidermolysis Bullosa is an inherited disease where the epidermis and basement membrane are not well attached due to mutations to any of the components of the hemidesmosome cell adhesion structure. Results in blister formation b/t basement membrane and epidermis –> skin fregility and severe scarring. A gene mutation of Collagen Type VII would cause the most severe form of Epidermolysis Bullosa.