Genetics 8

Question 0

Secondary Messengers

Answer 0

Soluble molecules that relay signals from activated receptors on the cell surface to target molecules inside the cell cytoplasm or nucleus.
Ex: cAMP, IP3, Ca2+

Question 1

Primary Messenger

Answer 1

Extracellular molecule (like a NT or hormone) that binds as a ligand to cell surface receptors to transduce the signal inside of the cell

Question 2

Effector

Answer 2

Molecule or protein that selectively binds to a protein that regulates its biological activity.

• The enzyme that produces the secondary messengers
• Ex: AC makes cAMP

Question 3

Multifunctional protein kinase

Answer 3

Kinases with more than one binding domain and more than one phosphorylation site.
-ex: cAMP-dependent protein kinase, Calicum/calmodulin protein kinase

Question 4

Autophosphorylation

Answer 4

Phosphorylates itself and for the most part, activates the kinase.
ex) Cam Kinase is fully active once it has autophosphorylated, which it is activated to do when Calcium/calmodulin binds to it.

Question 5

Phosphatase

Answer 5

Opposite function of kinase

-Dephosphorylates molecules.

Question 6

Trimeric G Proteins

Answer 6

Couple a receptor and an effector protein

• Both hormone/ligand and GTP are required for AC stimulation.
• Receptor agonists stimulate a low Km GTPase
• GDP and GTP modulate affinity of receptor to agonists, but not to antagonists (negative heterotropic allosteric interaction): part of cascade’s off mechanism.

Question 7

Agonist binding to receptor

Answer 7

Causes proper signaling pathway for an active downstream enzyme

Question 8

T/F, Antagonists occupy receptors, but do not yield a signal? Why or why not?

Answer 8

True, do not yield signal because they do not induce a conformational change that dissociates G protein’s alpha subunit.

Question 9

Why is the low Km GTPase important?

Answer 9

Having a low Km means half the Vmax is reached with small concentration of GTP.
-GTPase can hydrolyze GTP at low concentrations within the cytosol, which helps GTP bind when GDP dissociates from the alpha subunit.

Question 10

Alpha Subunit of Trimeric G protein

Answer 10

• Ga-GTPase: main GTPase function
• Dissociates from beta-gamma subunits when activated by receptor protein.
• 9 alpha subunit genes encode 12 different proteins
• Contains guanine binding site (GDP, GTP bind)

Question 11

Beta/Gamma subunit of Trimeric G protein

Answer 11

• Stay associated together

- Are involved in separate signaling pathway, impact other effector molecules.

Question 12

What happens to the alpha subunit when the receptor binds a ligand?

Answer 12

• Undergoes conformational change
• GDP it was holding onto dissociates
• low Km GTPase part of alpha grabs nearest GTP.

Question 13

What is the rate limiting step of G proteins?

Answer 13

• The dissociation of GDP from the alpha subunit.

- Provides key regulatory feature for activation of alpha subunit.

Question 14

Kinetics of GTPase activity

Answer 14

• In absence of ligand, kcat is 10 times faster than dissociation rate of GDP.
• This means that by default, the G protein is off (in its T form) most of the time.

Question 15

Gs

Answer 15

• Member of As subunit.
• Activates AC
• Tissue specific types of Gold and Ggust for sensory tissues.

Question 16

Go

Answer 16

• important in neurons with the activation of K+ channels.

- Most abundant in G protein subunit

Question 17

Gq

Answer 17

-Subunit to activate PLC-beta= phospholipase C-Beta

Question 18

Describe basic G protein Pathway from membrane receptor through generation of a 2nd messenger

Answer 18

• Trimeric G protein is associated with pm, GDP bound (= inactive form)
• Extracellular ligand binds to membrane receptor, causes conformational change within transmembrane receptor. H+R–> HR= activated
• Activated HR complex causes conformational changes in trimeric G protein for GDP to dissociate and GTP to bind to Gas.
• GTP binding dissociates Gas from HR and Gbetagamma. Hr–> T state, dissociates from ligand.
• Gas-GTPase travels and binds to effector (AC). Activates AC to produce cAMP
• Hydrolysis of Gas bound GTP inactivates AC.
• Inactive Gas dissociates from AC, reassociates with Gbetagamma subunits.

Question 19

Intracellular concentration of GTP is _____ compared to GDP

Answer 19

High

-GTP is more likely to associate with Gas

Question 20

What does GTP binding to Gas do?

Answer 20

1) Causes dissociation of G protein from Gbetagamma, to allow it to bind to the effector for the second messenger creation
2) Brings HR to T state to dissociate from ligand. Allows for cascade to be controlled based on presence of more ligands.
- More ligand to bind to the receptor? Transmits signal to continue activity.
- No more ligand? Receptor won’t be activated, cascade will stop.

Question 21

What activates AC activity?

Answer 21

Gas-GTPase going all the way back to the activated HR

Question 22

PKA

Answer 22

• Tetramer: 2 regulatory subunits and 2 catalytic subunits= inactive
• Regulatory subunits each have 2 cAMP binding sites, when cAMP binds, they dissociate from the catalytic subunits
• Peptide inhibitory site: has sequence that mimics protein to be activated by PKA

Question 23

Functional significance of a pseudo-substrate site on a regulatory subunit or domain

Answer 23

• 2 types of regulatory subunit proteins, differ in how well they mimic the substrate protein of the catalytic subunit.
• Type I: Pseudo P site- occupies and inhibits, but is not substrate.
• Type II: Auto P site- occupies catalytic site, is phosphorylated (an exact match to the site). Stays bound to inhibit until removed by cAMP binding.

Question 24

Peptide binding and catalytic regions mainly phosphorylate....

Answer 24

Serine or Threonine

Question 25

Phospholipase C Beta activated by?

Answer 25

Gq

Question 26

Beta of PLC is specific to cleaving?

Answer 26

PIP2, which can be cleaved into DAG, IP3, or Arachidonic Acid/Prostaglandin

Question 27

DAG-Protein Kinase C Pathway

Answer 27

DAG cleaved off by PLPc (other half is IP3) - Stays attached to membrane as transmembrane lipid - Once cleaved, it goes along the membrane to activate PKC

Question 28

IP3-Ca2+ Pathway

Answer 28

IP3 cleaved off by PLPc. - Becomes intracellular soluble molecule, very hydrophilic. - Once cleaved, binds IP3 dependent Ca2+ channels in ER to release Ca2+ into cytosol.

Question 29

PLPcbeta

Answer 29

-Gaq coupled PLC. Subtype specific for PIP2 cleavage from intracellular PM

Question 30

PLC-gamma

Answer 30

Activated by RPTK part of tyrosine kinase cascade

Question 31

PKC pathway of activation

Answer 31

1) PLC-beta cleaves PIP2 into DAG and IP3 2) DAG stays in membrane 3) IP3= soluble, binds Ca2+ in ER 4) Ca2+ released in cytosol 5) Ca2+ binds PKC in cytosol, increases its affinity to bind DAG in PM 6) Binding of Ca2+ and DAG to PKC activate it.

Question 32

Ca2+ pumps in the PM

Answer 32

1) Na+ driven Ca2+ exchanger: antiporter pumping Ca2+ out of the cell for every Na+ 2) Ca2+ pump: ATPase pumping Ca2+ out of cell.

Question 33

Ca2+ pumps in organelle membrane

Answer 33

- Keep Ca2+ out of cytosol, but high in organelle as a storage place 1) Ca2+ pump: ATPase pumps Ca2+ out of cytosol, into ER 2) Active Ca2+ importer in mit. 3) Ca2+ binding molecules in cytoplasm sequester Ca2+

Question 34

What are the sites of IP3 dependent Ca2+ Channel in ER membrane?

Answer 34

1) Low km activating site for Ca2+ 2) High km inhibitory site for Ca2+ (once it has been opened, or if cytoplasmic Ca2+ gets too high, Ca2+ binding will close the channel to stop the signal).

Question 35

AC is activated by what subunit?

Answer 35

Gas