GPCRs

Question 1

What are metabotropic receptors?

Answer 1

Indirectly linked to ion channels through signal transduction, such as G proteins.
This depend on multiple metabolic steps

Question 2

What are ionotropic receptors?

Answer 2

Direct exchange of ions through a pore
Made of 4/5 subunits

Question 3

What are the TM helices important for in GPCRs?

Answer 3

TM3 is next to the binding pocket, crucial for transduction of ligand binding
A conformational change between TM5 and TM6 allows the G-protein to bind

Question 4

How are platelets activated by GPCRs??

Answer 4

They are protease activated:
N-terminal is cleaved by thrombin which is part of the receptor (tethered ligand).
The G-protein binds to the C-terminus

Question 5

What subunits are guanine nucleotide-binding proteins made up of?

Answer 5

α, β and γ

Question 6

Outline a GPCR response up to the βγ subunit dissociating

Answer 6

1. Ligand binding to TM3, conformational change in receptor activates G protein.
2. TM5 and TM6 open their binding pocket, releasing GDP.
3. GTP binds and the α subunit dissociates from the βγ subunit.

Question 7

What stimulates GTPase activity?

Answer 7

RGS proteins (regulators of G-protein signalling)

Question 8

What are the functions of second messengers?

Answer 8

Activity is controlled by G-proteins
Carry signals inside a cell-
-Hydrophobic lipids in the membrane
-Small soluble molecules in the cytoplasm
-Ca2+

Question 9

How do cholera toxins affect cells?

Answer 9

Add ADP ribose to Gsα subunits, preventing it switching off.
This leads to adenylyl cyclases producing lots of cAMP and activating kinases.
Cl-, Na+ and H2O are secreted, leading to excess fluid in the small intestine

Question 10

How does Bordetella pertussis affect cells?

Answer 10

ADP ribose binds to Giα subunits, inactivating them. cAMP is increased and the respiratory epithelium is degraded. Mucus is discharged, triggering coughing fits

Question 11

Give some examples of activating mutations with GPCRs?

Answer 11

Hypoparathoidism- Parathyroid Ca2+ sensor
Night blindness- Rhodopsin
Thyroid cancer- Thyroid hormone receptor

Question 12

Give some examples of loss of function mutations with GPCRs?

Answer 12

Colour blindness- cone cell opsin
Hyperthyroidism- Parathyroid Ca2+ sensor
Retinal degeneration- rhodopsin
Diabetes inspidus- vassopressin receptor

Question 13

How are 90% of uveal melanoma cases caused?

Answer 13

Mutations in Gqα, blocking GTP hydrolysis. This causes permanent signal transmission and constituitive growth pathways- cancer.

Question 14

Outline cAMP

Answer 14

Synthesised by adenylate cyclase
10 isoforms
C1a and C2a on the C-terminus are required to activate the GPCR

Question 15

Outline a general Gs protein coupled response

Answer 15

1. The ligand binds to the receptor and activates the G-protein
2. α subunit dissociates from the βγ and binds adenylate cyclase
3. The activated enzyme catalyses cAMP production
4. cAMP activates protein kinase A
5. PKA phosphorylates a protein which imitates a response in the cell

Question 16

How are G-proteins switched off?

Answer 16

1. Agonist dissociates from the receptor
2. GTPase activity of Gαs
3. cAMP is broken down by phophodiesterase
4. Enzymes are phosphorylated

Question 17

What synthesises cGMP?

Answer 17

guanylate cyclase

Question 18

How do second messengers determine the specific message in a cell?

Answer 18

Concentration and frequency of conc changes.
Concentration of second messengers is determined by rates of production, diffusion and removal (by phosphodiesterases)

Question 19

What is phopholipase Cβ?

Answer 19

Second messenger and also effector of Gq subunit.
Cleaves phosphatidyl inositol (PIP2)
Produces IP3 which binds to calcium channels in the ER
DAG is hydrophobic and remains in the membrane, activating PKC

Question 20

What sort of kinases are phosphokinase Cs?

Answer 20

Ser/Thr kinases

Question 21

What activates PKCs?

Answer 21

DAG (C1 domain)
Ca2+ (C2 domain)

Question 22

How are PKCs activated in research?

Answer 22

A DAG analogue called PMA phorbol ester is used

Question 23

How is negative feedback created with phospholipase Cβ.

Answer 23

PKC phophorylates PLCβ

Question 24

What does Ca2+ usually control in cells

Answer 24

Synaptic transmission
Activating T-lymphocytes
Hormone synthesis/secretion
Fertilisation
Muscle contraction
Cytokinesis

Question 25

How are ER calcium levels maintained?

Answer 25

A store operated channel made of ORAI and gated by a STIM dimer

Question 26

How can loss of function mutations of ORAI affect an organism?

Answer 26

Severe combined immunodeficiency (SCID) due to impaired Ca2+ release

Question 27

What can overstimulation of GPCRs lead to?

Answer 27

Tachyphylaxis and cancer

Question 28

What can rhodopsin overstimulation lead to?

Answer 28

Prolonged photon response and rod apoptosis

Question 29

How can GPCRs be inactivated?

Answer 29

GPCR kinases (GRKs) and β-arrestin can stop G-protein binding or internalise a receptor for degradation and recycling

Question 30

How would a short piece of genetic information be statistically studied?

Answer 30

1. mRNA would be extracted from tissue and converted to cDNA
2. A sequencing library containing cDNA molecules in each sample is prepared
3. Sequence on an Illumina NGS machine
4. Run pipeline steps to make comparisons

Question 31

How are similarities in RNA-seq found?

Answer 31

UMAPs. Each dot is a cell, the closer they are, the more similar. This gives insights into genes expressed, cell lineage and tissue composition changes

Question 32

How are disease associated genes found?

Answer 32

Genome wide association study (GWAS)
Finds SNPs in the genome that are more frequent in patients than healthy individuals