receptors and signalling Flashcards
what is the role of receptors?
receive extracellular information via ligands, and initiate a response in the cell
list the key differences between cell surface receptors and intracellular receptors
cell-surface receptors:
hydrophilic ligand, can be large in size, located on the cell surface. generally consist of three topological domains; extracellular domain, transmembrane domain, and intracellular domain
intracellular receptors:
hydrophobic ligand, small, can be in the nucleus, consist of DNA binding domain, ligand binding domain. gene has receptor-binding element
what is adrenaline?
small but hydrophilic molecule derived from tyrosine. originates in the adrenal gland. is responsible for increase in blood pressure, heart rate, and metabolism
what is glucagon?
29AA peptide that originates from a-cells of the pancreas. stimulates glucose synthesis, glycogen and lipid breakdown
what is insulin?
a peptide complex consisting of an A chain and B chain. originates from B cells of the pancreas, stimulates glucose uptake, protein and lipid synthesis in the liver and muscle cells
what is retinoid acid receptor an example of?
a DNA binding protein that works as a ligand-activated transcription factor
what do ligand-activated transcription factors do?
bind to their respective intracellular receptor protein and alter the ability of these proteins to control the transcription of certain genes
how are ligand-activated transcription factors made soluble?
binding to a specific carrier protein from which they dissociate before entering the target cell
how does ligand binding activate ligand-activated transcription factors?
inactive receptors are usually bound to inhibitory protein complexes, ligand binding causes a conformational change which causes the inhibitory protein to dissociate whilst causing the receptor to bind co-activator proteins that stimulate gene transcription
how can you identify the receptor for a ligand of interest?
isolate proteins that bind a ligand from a cell lysate by using ion-exchange chromatography
what is the basis for ion-exchange chromatography?
proteins are separated according to charge by packing the column full of small beads carrying a positive or negative charge, this allows proteins to be fractionated according to the arrangement of charges on their surface
how can a radioisotope labelled ligand be used to identify a receptor?
1) perform ion-exchange chromatography on DEAE cellulose using radiolabelled ligand
2) perform additional purification using SDS-PAGE
3) using this fraction, obtain partial peptide sequences via mass spectrometry for the receptor
4) use WGS data to search database and identify receptor gene
what is the benefit of obtaining a cDNA clone?
- allows production of recombinant proteins and conduct DNA binding assays
- produce GFP-tagged proteins to observe cellular localisation
- introduce various point mutations to examine importance of each amino acid in each functional property
