GPCRS

Question 1

Outline the Discoveries of John Langley in the 19th and early 20th Century.

Answer 1

1st: He anaesthetized fowl and found injection with nicotine caused contractions in leg muscles in the denervated leg. By injection with curare, the contraction would be stopped.
2nd: He injected curare to the fowl and found direct electrical stimulation still produced contraction

Question 2

What conclusions did Langley draw from the 2 key Experiments?

Answer 2

• Nicotine acted directly on the muscle cell

- Curare acted on an accessory substance of the muscle cell called the receptive substance

Question 3

What was Paul Ehrlich responsible for?

Answer 3

He introduced the term receptor in 1905 suggesting their roles to have specific tasks and binding tasks.

Question 4

Outline the findings of A.V. Hill in 1909

Answer 4

He was the first to quantitatively express the receptor idea in terms of a bimolecular reaction (nicotine and a receptor).

Question 5

What experiments of A.V. Hill led to this finding?

Answer 5

He studied the time of the contraction of a frog muscle produced by nicotine. He showed the equilibrium concentration-effect curve fitted his equation.

Question 6

What is the Hill equation?

Answer 6

y = N/ (k1 + KN) - M

y = response height
N = nicotine concentration
k1 and k = constants
M = threshold

Question 7

What experiments did Clark and Gaddum responsible perform?

Answer 7

Gaddum: studied the action of adrenaline and ergotamine of the rabbit uterus
Clark: studied the action of Ach and atropine on frog heart

Question 8

What did Clark and Gaddum first introduce?

Answer 8

The log[concentration] vs effect curve.

Question 9

Who termed the idea of a “Second Messenger”?

Answer 9

Earl Sutherland 1950s- 60s. Used biochemical techniques to show that an intermediate substance was needed for the action of adrenaline. The substance was cAMP

Question 10

What is the name of the enzyme responsible for the production of cAMP?

Answer 10

Adenylyl Cyclase.

Question 11

What were N proteins and what led to their discovery?

Answer 11

Rodbell and Birnbaumer in the late 1960s observed that activation of adenylyl cyclase required proteins that bound GTP as transducers- they called these proteins N proteins

Question 12

Who coined the term signal transduction?

Answer 12

Rodbell. He proposed a three step model in which the N proteins serve as a switch to mediate against receptor interactions

Question 13

Outline the principles behind the technique of Radioligand Binding.

Answer 13

Its a method used to characterise receptor and determine their anatomical distribution. The ligand is radioactively labelled (often tritium). This method was famously used by Lefkowitz in 1970s

Question 14

What were the advantages of Radioligand Binding?

Answer 14

• led to rapid progress in understanding regulation of receptors
• led to rapid expansion of discovery in receptor subtypes
• proved useful for developing and exploring new theories of receptor action.
• allowed for the first time an approach to isolation of GPCRs

Question 15

What are the 3 parts of the Ternary Complex Model?

Answer 15

• the extracellular agonist
• the transmembrane GPCR
• the intracellular G-protein

Question 16

Who proposed the Ternary complex model?

Answer 16

Lefkowitz 1980. He proposed it as a general mechanism for receptor activation

Question 17

What does Receptor signalling rely on following the principles of this model?

Answer 17

Signalling relies on an ALLOSTERIC MECHANISM.

• allosteric coupling is mutual between intracellular G-protein and extracellular ligand
• binding of ligand to receptor increases affinity of receptor for G-protein while binding of G-protein increases affinity of receptor to ligand.

Question 18

What were the findings of Lefkowitz and Caron in 1976 regarding the B-adrenergic receptor?

Answer 18

They stabilized and purified the receptor from frog cells. They found each receptor was made of a single polypeptide chain, all were glycoproteins 60 -65 kDa in weight.

Question 19

What were the key findings following the cloning of the B-adrenergic receptor in 1986?

Answer 19

Lefkowitz and others: the cloned receptor had seven transmembrane helices as well as high similarity to the rhodopsin receptor sequence.

Question 20

How was the finding that all GPCRs have 7 transmembrane regions corroborated?

Answer 20

Shosaku Numa: he sequenced the 3rd GPCR which was the muscarinic Ach receptor in 1986.

Question 21

Name the three ways in which a receptor is ale to bind to specific ligands?

Answer 21

• sequences in intracellular loops determine G-protein interaction specificity
• Ligand binding is determined by sequences in the extracellular loops and/or N-terminus depending on the class of the ligand.

Question 22

What was the first insight into structural changes in GPCRs following activation?

Answer 22

Hubbell and Khorana found that motions in helix 6 of rhodopsin was important in the activation mechanism. They found this using Electron Paramagnetic Resonance (EPR)

Question 23

Outline the principles behind EPR.

Answer 23

Its a technique for studying molecules with unpaired electrons as these have unbalanced spins. It follows the same principle as NMR but electrons spins are excited not protons.

Question 24

Name 3 key analytical techniques used in elucidating the structural changes in GPCRs.

Answer 24

• IR
• NMR
• Mass Spectroscopy
  These were performed primarily on rhodopsin and the beta-adrenergic receptors.

Question 25

What were the findings regarding structural changes in GPCRs.

Answer 25

Small change in the extracellular region - binding of agonist- results in a large change in the intracellular region. Specifically, there is a displacement of Helix 6 and the opening of a hydrophobic cleft where the G-protein will bind

Question 26

Name the two families of proteins which regulate GPCRS

Answer 26

Beta-arrestins and G-protein coupled kinases (GRKs)

Question 27

Outline the process by which a GPCR can be desensitized.

Answer 27

• the GRK phosphorylated the GPCR often on the carboxyl terminal (intracellular)
• phosphorylation promotes the interaction of B-arrestins with receptor
• B-arrestins desensitize the receptor