cell signalling and nuclear receptors

Question 1

what are the characteristics of cell surface receptors?

Answer 1

• hydrophilic ligand
• can be large
• consist of extracellular domain, TM domain and intracellular domain

Question 2

what are the characteristics of intracellular receptors?

Answer 2

• hydrophobic ligand
• small molecules
• can be in the nucleus
• consists of DNA binding domain, ligand binding domain, co-activator domain

Question 3

give an example of a ligand for a cell surface receptor

Answer 3

adrenaline - small, hydrophilic, derived from tyrosine and originates from adrenal medulla

Question 4

what is the shared function of all nuclear receptors?

Answer 4

all bind to their respective intracellular receptor proteins and alter the ability of these proteins to control the transcription of certain genes

Question 5

how does ligand binding alter the nuclear receptor?

Answer 5

causes a conformational change that causes inhibitory proteins to dissociate whilst causing the receptor to bind co-activator proteins that stimulate gene transcription

Question 6

what was the first nuclear receptor to be discovered and when was it discovered?

Answer 6

glucocorticoid receptor identified by cDNA cloning in 1985

Question 7

how can receptors for a ligand of interest be identified?

Answer 7

proteins from a cell lysate can be isolated using ion-exchange chromatography, SDS-PAGE and bioinformatic analysis of fragments

Question 8

what is the principle behind ion-exchange chromatography?

Answer 8

• proteins are separated according to charge
• uses a column packed full of small beads carrying a positive or negative charge, allowing proteins to be fractionated according to the charge on their surface

Question 9

when performing chromatography, why is absorbance often measured at 280nm?

Answer 9

due to the presence of aromatic amino acids such as tyrosine, tryptophan and histidine

Question 10

before WGS was available, how was gene sequence fora receptor identified?

Answer 10

• using fraction from ion-exchange chromatography, animals injected with peptides
• anti-GR antibodies were generated
• cDNA clones could be isolated

Question 11

what assays are permitted by obtaining cDNAs?

Answer 11

• production of recombinant proteins + DNA binding assays
• GFP tags to observe cellular localisation
• introduction of point mutations to understand properties of protein
• generate chimeric receptors