Previously in Cell Bio

1. Previously in Cell Bio Signals are detected via binding interactions Binding interactions governed by protein folding Protein folding dictated by amino acid sequence Hypotheses for ‘problem’ in Graves’ Disease Positive signals (TRH or TSH) altered to increase amount or affinity for their receptor Signal receptor altered to increase their affinity for hormone Problem with signal relay ‘inside’ thyroid

2. TSH Receptor: Extracellular Plasma membrane Cytosol TSH Receptor:from “The Thyroid Manager” Ch16

3. Graves’ hypothesis 1: TSH, TSH-Receptorinteraction ‘too strong’ According to this hypothesis and what we now 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.

4. Blood tests show T3 and T4 levels are elevated TSH levels are decreased TSH/TSH receptor interactions have same binding constant vs. normal. Therefore: Perfectly logical hypothesis…….

5. 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? Notsupported by data Now what?

6. 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

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

8. Thyroid plasma membrane is barrier • to polar molecules Even more • 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?

9. 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

10. Peripheral Membrane proteins Characteristics • 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.

11. Membranes and membrane proteins How can a polar signal gain access to the cytosol • Direct access: From the ‘outside’ • Pores • Channels • Pumps • From cytosol to cytosol • Gap junctions

12. Membrane proteins Indirect access: Receptors If signaling molecule never gains access to cytosol how can the information be transmitted? Extracellular domain Plasma Membrane Cytoplasmic Domain TSH Receptor:from “The Thyroid Manager” Ch16

13. 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 cause overproduction of T3 and T4 How could you test your hypothesis?

14. 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

15. Other mechanisms that regulate protein function • Compartmentalization • Change in rate of synthesis Common traits? • Cleavage • Phosphorylation/dephosphorylation Common traits?

16. 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?

17. 2nd Messengers and Signaling Cascades Getting the signal to where it needs to go