Previously in Cell BioSignals are detected via binding interactionsBinding interactions governed by protein foldingProtein folding dictated by amino acid sequence

Hypotheses for ‘problem’ in Graves’ DiseasePositive signals (TRH or TSH) altered to increase

amount or affinity for their receptorSignal receptor altered to increase their affinity

for hormoneProblem with signal relay ‘inside’ thyroid

TSH Receptor:

TSH Receptor: from “The Thyroid Manager” Ch16

Plasma membrane

Extracellular

Cytosol

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__?___ binding constant 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 ‘overacting’•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

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

-Protein-protein-Protein-phospholipid head

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

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

Check out CBI 3.9 and 3.8/(Hemoglobin) Chime models to see examples

Other mechanisms that regulate protein function

•Compartmentalization•Change in rate of synthesis

Common traits?

•Cleavage•Phosphorylation/dephosphorylation

Common traits?

Receptor’s role (summary)

Able to transduce signal because of:•Placement in membrane (span it)•Ability to bind ligand

•Ligand -induced conformational changes

So the signal ‘gets in’ without physically crossing membrane

BUT How do you go from a shape change to causing a change in gene expression?

2nd Messengers and Signaling Cascades

Getting the signal to where it needs to go