L14 Membrane Protein Function

How does signal transduction (cellular signaling) occur?

1. binding of signaling molecule x receptor

2. relay primary message (hormone) to cell interior, by generation of secondary messenger (cAMP)

3. signal amplification & transduction

4. response

What’s the structure of GPCRs? What effect it has after bound?

• has 7 TM segments

• conformational change releases G-proteins → propagate intracellular signaling cascade → cAMP production → hydrolyze GTP to GDP, terminating activity

natural: serotonin, epinephrine, prostaglandins, dopamine, psilocin/cybin

synthetic: morphine, histamine, LSD

what influences binding affinity of AA side chains / functional groups? What value indicates binding affinity?

• non-covalent interactions

• Kd value - dissociation constant

  • ⬇Kd = stronger binding

  • Kd = [A][B]/[AB] = K off / K on

    • [A][B] = free molecules; [AB] = bound complex

      • ⬆[AB] = ⬇️Kd

    • K off = dissociation rate constant; K on = association rate constant

• binding is saturable based on stoichiometry; reversible for non-covalent interactions

After β2-Adrenergic Receptor is bound, what happens?

Ligand binding → 2A inward movement of extracellular TM5 → signal transmitted inside by 14A outward movement of TM6 → release of Gα-GTP → activates adenylyl cyclase to produce cAMP → activates PKA (transferase), phosphorylates other enzyme

• is a GPCR

Enzyme-linked Receptors:

• what’s the structure?

• how does it work?

• contain 1TM that may be homodimer - dimerize upon ligand binding

• binds hormone outside → induce conformational change → allow enzyme to work inside

• activation leads to auto-phosphorylation / phosphorylation by tyrosine kinase

  • auto = one subunit phosphorylate another

• e.g. insulin, EGF, Jak/STAT

Phospholipid-mediated Signaling

• how does it work?

Phospholipases hydrolyze phospholipids to produce other secondary messengers, leading to Ca²⁺ release from ER

Which are the competing hormones? How do they compete?

insulin & epinephrine

• insulin is stronger, always win out

• insulin ON:

  • phosphorylation of insulin receptor substrate (IRS-1), activating PKB → phosphorylates β-adrenergic receptor

  • leading to internalization & degradation of β-adrenergic receptor, terminating GPCR signaling → no epinephrine signaling

what kind of molecule can pass membrane by passive diffusion?

• small, uncharged/lipophilic molecules

• nutrient in, inorganic ions (wastes) out

Rank permeability across membrane from most to least permeable:

• Oxygen, Alanine, H₂O, H⁺

1. Oxygen - small, uncharged

2. H₂O - uncharged, polar

3. H⁺ - charged

4. Alanine - has 2 charged functional groups, more polar

What type of curve does facilitated diffusion have? why?

• hyperbolic

• is saturable (when all binding sites on membrane proteins are occupied)

GLUT transporters

• what type of diffusion?

• what is is triggered by?

• how does it work?

• glucose binding → conformational change

• transport is concentration dependent & saturable

Beta barrel proteins

• what is it composed of?

• what is the structure?

• integral membrane protein

• composed of β-strands forming pore in membrane

• inside (AA facing) = hydrophilic (polar pore); outside = hydrophobic

• need less AA to span bilayer (β-strands are more extended)

• allow passive movement

Channel proteins

• what does it transport?

• what are its important features?

• facilitate diffusion/transport of charged ions

• important features: selectivity, rapid conductance, can be stimuli-gated

Potassium ion channel

• what processes is it important for?

• what’s the structure & their function?

• how does gating work?

• cell volume regulation, hormone secretion, electrical impulse formation

• each subunit contributes to selectivity filter of 5 AAs (TVGYG) - contribute to K⁺ binding

• 4 backbone carbonyls C=O & Thr side chain -OH bind K⁺ ions

• changing sequence alters selectivity for other cations

• gating:

  • response to specific stimuli (voltage/pH change) → helix bending at conserved Gly (in regulatory domain)

  • Gly99 = molecular hinge to open/close gate

How does molecules move in Active Transport?

what is Primary active transport?

what is secondary active transporters?

• move against concentration gradient

• Primary active transport = ATP breakdown, light energy, e^- passing → generates energy for transport (Ca²⁺-ATPase, Flippase & Floppase)

• Secondary active transporters = uses gradient of one molecule to power formation of another (Na⁺-glucose transport)

  • antiporter, symporter