Previously in Cell Bio1) Signals are detected via binding interactions

2) Strength of binding interactions can be calculated– referred to as the dissociation constant

Kd= off/ on @ equilibrium = [R][L] [RL]

3) Binding interactions governed by protein folding

4) Protein folding dictated by amino acid sequence

Can we predict protein structure?Motifs and Domains

How do you change a protein’s shape?Alter the chain-- modificationsChange the local environment–

what it is floating in or binding with

TSH Receptor: What level of structure?

TSH Receptor: from “The Thyroid Manager” Ch16

Plasma membrane

Extracellular

Cytosolic

Graves’ hypothesis 1: TSH, TSH-Receptorinteraction ‘too strong’

According to this hypothesis and what wenow know about protein binding……

T3 and T4 levels should be _?_ in Graves’vs. normal.

TSH levels should be __?__ in Graves’ vs. normal

TSH/TSH receptor interactions should show__?___ Kd vs. normal.

Blood tests show

T3 and T4 levels are elevatedTSH levels are decreasedTSH/TSH receptor interactions have same

binding constant vs. normal.

Therefore: Perfectly logical hypothesis…….

Now what?

Not supported by data

Hypothesis 2: Mutation in signaling within cell leading to increase in thyroid hormone production

Normal activation is the result of signal transduction second messenger cascade

How does signal transduction work?What could have gone wrong?

What do we know so far?•Thyroid is ‘over reacting’•Pituitary normally responsible for thyroid

stimulation through levels of TSH

•Graves’ patients have normal/decreased levels of TSH in blood

•Binding affinity between TSH and TSH-R normal

More of what we know•TSH is water soluble hormone (why is this important?)

Figure 4-1. Schematic drawing of human TSH, based on a molecular homology model built on the template of a hCG model14. The a-subunit is shown as checkered, and the b-subunit as a solid line. The two hairpin loops in each subunit are marked L1, L3; each subunit has also a long loop (L2), which extends from the opposite site of the central cystine knot. The functionally important a-subunit domains are boxed. Important domains of the b-subunit are marked directly within the line drawing (crossed line, beaded line and dashed line): For further details the reader is referred to Grossman et al.2. (Reproduced from Grossman,M, Weintraub BD, SzkudlinskiMW-Endocrin Rev (4) 18:476-501,1997, with permission of the Endocrine Society).

From “The Thyroid manager”

Even more•Thyroid plasma membrane is barrier to polar molecules

•TSH interacts with a receptor on the surface of thyroid cells

HOW and WHY is the thyroid responding as though over-stimulated?

And to get to the answer of that question: How do signals get passed across membranes?

Characteristics of Transmembrane Proteins

•Hydrophobic face of protein in transmembrane region-one continuous structure or multiple regions of 2° structure

•Charges ‘anchor’ transmembrane region

•Asymmetric orientation

Peripheral Membrane proteinsCharacteristics

•Associations with membrane not as strong•Various means of attachment

-Protein-protein-Protein-phospholipid head- Lipid modification imbedded into membrane

Fig 3-32 Molecular Cell Biology by Lodish et al. 5 th ed

Membranes and membrane proteinsHow can a polar signal gain access to the cytosol

Direct access: From the ‘outside’•Pores•Channels•PumpsFrom cytosol to cytosol•Gap junctions

Membrane proteinsIndirect access: Receptors

TSH Receptor: from “The Thyroid Manager” Ch16

Extracellular domain

Cytoplasmic Domain

Plasma Membrane

If signaling molecule nevergains access to cytosol how canthe information be transmitted?

Transmembrane receptors•Same general structure as other transmembrane proteins•Able to bind specific ligand•Ligand binding causes conformational change

What change in the TSH receptor could causeoverproduction of T3 and T4

How could you test your hypothesis?

Allosteric transitions

What are they, why are they important, How do they relate to signal transduction

•R T state transitions

•Cooperative binding

Examples DNA helicase and ras (links from index page)

2nd Messengers and Signaling Cascades

Getting the signal to where it needs to go

For Tuesday: summarize a cascade involving. 1) cGMP 2) RTK 3) IP3 (inositol triphosphate) 4) Ca++ 5) DAG (diacyl glycerol)

Email me one paragraph summary of how that onecascade works by midnight Monday (think ‘handout’)