Lecture 14: Membrane Protein Function

Question 1

Cellular Signaling

Answer 1

• All processes involve biochemical interactions, allowing a cell to carry out its function
• Binding of signaling molecules to receptors initiate processes such as metabolic pathways and gene expression
• Proteins essential for carrying out the response

Question 2

Signal Transduction

Answer 2

• Binding specifically to a signalling molecule in response to a stimulus
• Reception of message by receptor(integral membrane protein)
• Relay of primary message to cell interior by intracellular secondary messenger
• Amplification and transduction of signal
• Response and termination of signal

Question 3

G-Protein-Coupled Receptors

Answer 3

• GPCRs contain 7 TM segments
• Conformational changes release the G protein
• Can bind to many different ligands
  
  • Natural: Serotonin, Epinephrine, Prostaglandins, Dopamine, Psilocybin
  • Synthetic: Morphine, Histamine, LSD

Question 4

Characterizing Binding Interactions

Answer 4

• Non-covalent interactions betwen amino acid side chains and molecules functional groups influence binding affinity
• Binding affinities used to characterize and compare non-covalent interactions between two biomolecules
• Lower Kd equals stronger binding

Question 5

B- Adrenergic Receptor

Answer 5

• Ligand binding induces small changes in TM5 on extracellular side
• 14 A movement in TM6 transmits the signal inside
• Major conformational changes in TM6 promote release of Ga-GTP, which activates adenyl cyclase to release cAMP. cAMP activates Protein Kinase A, which phosphorylates and activates/inactivates other enzymes

Question 6

Ras Protein

Answer 6

• Member of superfamily of small GTPases that bind and hydrolyze GTP
• Activated in signalling pathways that initiate cell proliferation and apoptosis
• A conformational change seen in switch 1 and 2 motifs upon phosphate release(GTP -> GDP)
• Defects in GTP hydrolysis can lead to cancer

Question 7

Signalling and Human Health

Answer 7

• Defects in the pathway can lead to disease
• PTMs and conformational changes play key role in the pathway
• Mutations in the receptors or effector proteins can prevent ligan-receptor interactions needed for activation/inactivation
• Drugs can be designed to bind and inhibit proteins involved in signalling to modulate cellular response

Question 8

Other types of signalling

Answer 8

• Enzyme-linked receptors: Usually contain a single TM segment that may dimerize upon ligand binding
  eg. Insulin, epidermal growth factor, Jak/STAT
• Activation leads to phosphorylation by tyrosine kinases
• Phospholipid mediated Signalling: Phospholipases hydrolyze phospholipids to produce secondary messengers like diacylglycerol(DAG) or IP3 leading to release of Ca from the ER
  
  • eg. Eicosanoid and AKT signalling

Question 9

Hormone vs Hormone

Answer 9

• Insulin and epinephrine are competing hormones
• Phosphorylation of Insulin Receptor Substrate(IRS-1) and activation of pathway leads to phosphorylation of B-adrenergic receptor by Protein Kinase B
• PTM leads to internalization and degradation to terminate GPCR signaling, epinephrine can no longer bind

Question 10

Membrane Transport

Answer 10

• Small, uncharged or lipophilic molecules can cross membrane through passive diffusion
• Nutrients go in - go out
• Inorganics ions in and out of
• Integral membrane proteins are important for transport
  
  • Facilitated diffusion
  • Active transport(w or wo ATP)

Question 11

Facilitated Diffusion is Saturable

Answer 11

• Facilitated transport is dependent on the presence on binding sites on membrane proteins
• The rate of transport is saturable at high substrate concentration
• A hyperbolic curve is similar to what is seen for catalytic enzymes

Question 12

Channel Proteins

Answer 12

• Membrane transporters that facilitate diffusion are known as ion channel proteins
• The structure of the membrane protein is key for its function
• Important features of ion channels
  
  • Selectivity
  • Rapid conductance of ions
  • Can be gated due to stimuli

Question 13

Potassium Ion Channel

Answer 13

• Essential for many cellular processes
  - Regulation of cell volume
  - Secretion of hormones
  - Electrical impulse formation
• Each subunit contributes to a selectivity filter of 5 amino acids that contribute to K+ binding
• 4 backbone carbonyls and Thr side chain hydroxyl binds K+ ions
• Changing sequence alters selectivity for other cations

Question 14

Gating the Potassium Channel

Answer 14

• In response to a specific stimuli, helix bending at a conserved Gly residue occurs at regulatory domain
  
  • Gly99 acts as hinge to open/close gate

Question 15

Active Transport

Answer 15

• Active Transport is movement of molecules against concentration gradient
• In primary active transport, the breakdown of ATP or passing of electrons generates energy for transport
• Secondary transporters use gradient of one molecule to power formation of another