Lecture 11

Question 1

What are G-protein coupled receptors?

Answer 1

large and diverse family with a common strucure of seven membrane spanning alpha helices and an internal ligand-binding pocket that is specific for particular ligands

Question 2

GPCR

Answer 2

G-protein coupled receptors

Question 3

What are GPCR’s coupled to?

Answer 3

heterotrimeric G proteins

Question 4

What do heterotrimeric G proteins contain?

Answer 4

three subunits designated alpha, beta and gamma
- Galpha subunit is a GTPase switch protein that alternates between an active (“on”) state with bound GTP and inactive (“off”) state with bound GDP. The “on” form separates from the β and γ subunits and activates a membrane-bound effector.
- The β and γ subunits remain bound together and can also transduce signals

Question 5

What does ligand binding cause in the GPCR?

Answer 5

conformational change in certain membrane-spanning helices and intracellular loops of the GPCR, allowing it to bind to and function as a guanine nucleotide exchange factor (GEF) for its coupled Gα subunit, catalyzing
dissociation of GDP and allowing GTP to bind. The
resulting change in the conformation of the switch region in Gα causes it to dissociate from the Gβγ subunit and the receptor
and interact with an effector protein

Question 6

What is GEF

Answer 6

guanine nucleotide exchange factor

Question 7

What do FRET experiments demonstrate?

Answer 7

demonstrate receptor-mediated dissociation
of coupled Gα and Gβγ subunits in live cells

Question 8

What are the proteins activated/inactivated by G proteins?

Answer 8

the effector proteins activated (or inactivated) by heterotrimeric G proteins are either enzymes that form second messengers (e.g., adenylyl cyclase, phospholipase C) or ion channels

Question 9

What determines the function of the G protein?

Answer 9

The Galpha subunit

Question 10

What does the hormone epinephrine do?

Answer 10

which mediates the fight-or-flight response

Question 11

What does the hormone epinephrine bind to?

Answer 11

multiple subtypes of GPCRs in multiple cell types, with varying physiological effects

Question 12

What did efforts to identify orphan GPCRs lead to the discovery of?

Answer 12

orexins, hormones that regulate feeding behavior and sleep in both animals and humans

Question 13

What do membrane receptors bind?

Answer 13

hydrophillic signalling molecules

Question 14

What do receptors activate?

Answer 14

G-proteins which transduce the signal

Question 15

What are the 4 common elements that GPCR signal transduction pathways share?

Answer 15

*(1) A receptor that contains seven membrane-spanning α helices
*(2) A receptor-activated heterotrimeric G protein cycling between GTP-active and GDP-inactive forms
*(3) A membrane-bound effector protein
*(4) Proteins that desensitize the signaling pathway

Question 16

What do many signal transduction pathways have?

Answer 16

*Involve second messengers
*Have short-term effects in the cell – rapid activation/inhibition of existing proteins, including enzymes or ion channels

Question 17

What is the general structure of GPCRs?

Answer 17

*7 transmembrane alpha-helical regions (H1-H7)
*N-terminus on outside of cell (hydrophillic), C-terminus on cytosolic face (interacts with G-switch)
*ligand binding site located in a groove located in the middle of the plane of the membrane
*ligand interacts non-covalently with specific amino acids in the interior-facing side of several membrane spanning alpha-helices

Question 18

How GPCRs are there for the human genome?

Answer 18

800

Question 19

What do all GPCR’s have?

Answer 19

*The same orientation in the membrane – N-terminus outside, C-terminus in cytosol
*Contain seven transmembrane α-helical regions (H1–H7)
*Have four extracellular segments (E1–E4)
*Four cytosolic segments (C1–C4)
*Ligands have not been identified for many “orphan” receptors

Question 20

What is the structure of the β2-adrenergic receptor in the inactive state and in the active state with a bound ligand and with its associated heterotrimeric G protein, Gs?

Answer 20

*Ligand binding converts β2-adrenergic receptor into a conformation that binds its trimeric G protein.
*(a) (–) Ligand
*β2-adrenergic receptor – no ligand bound – inactive state
*Inactive trimeric G protein – Gs: Gαs (dark purple), Gβ (light purple), and Gγ (pink) – unable to interact with inactive receptor.
*(b) (+) Ligand
*Ligand binding causes movement of the receptor TM5 and TM6 helices and changes in the C3 loop to create a surface that forms extensive interactions parts of Gαs
*Gαs small conformational change
*Lengthening the α5 helix creates a large surface consisting of the N-terminal α-helical segments αN and α5 that bind mainly to receptor TM5 and
*TM6.
*Triggers release of GDP and Gβγ
*(c) Binding of epinephrine to the side chains of multiple amino acids in the interior of the β2-adrenergic receptor

Question 21

How are the G alpha and G gamma subunits of GPCR’s attached to the membrane?

Answer 21

covalently attached lipids

Question 22

What does the binding of the ligand to the GPCR lead to?

Answer 22

conformational change of receptor which allows the binding of the G alpha subunit, receptor binding laters conformation of G alpha subunit causing release of bound GDP

Question 22

What is the general mechanism for the activation of effector proteins associated with GPCR’s?

Answer 22

*Step 1: Ligand binding induces receptor activation conformational change.
hydrophobic proteion of G protein holds it on the inner side of the membrane
*Step 2: Activated receptor binds to trimeric G protein.
*Step 3: Activated receptor GEF activity stimulates Gα subunit release of GDP.
*Step 4: GTP binding changes Gα conformation
*Dissociates Gβγ (Gβγ subunit activates other effector enzymes in some pathways (b))
*Activates Gα
*Step 5: Gα·GTP activates effector enzyme.
*Step 6: Gα intrinsic GTPase activity hydrolyzes GTP to GDP – dissociates Gα and turns off effector enzyme. (G protein active for minutes or less)

Question 23

What do ligand activated GPCR’s catalyze?

Answer 23

Ligand-activated G protein–coupled receptors – (GEFs) catalyze exchange of GTP for GDP on the α subunit of a heterotrimeric G protein

Question 23

What is the box in the mechanism of the activation of effector proteins?

Answer 23

Box – trimeric G protein Gα and Gβγ subunits – tethered to the membrane by covalently attached lipid molecules