Brain Metabolism
Molecular Mechanisms
Underlying Brain Function
Dr Eve Lutz
Department of Bioscience
Lectures 3 & 4
Molecular Biology of Receptors
Lecture notes last updated
28/12/2003
Introduction
Determining Receptor Structure
- Primary amino acid sequence
- Protein purification
- Isolation of the gene
and sequence determination
- Expression cloning
(antibody probes, ligand binding, other functional assay)
- Homology cloning
- Silicon cloning
Identifying receptor function
- Blast search sequence
to identify receptor type or family
- Determine localisation
- Determine ligand specificity
- Heterologus expression
- Xenopus oocytes
- Eukaryotic cell
line (COS, HEK 293)
- Characterise intracellular
events which occur following receptor activation
Receptor domains - key features
- Intramolecular interactions
- Docking sites for ligands
- Docking sites for G
proteins
- Docking sites for kinases/phosphatases
- Docking sites for adaptor/scaffolding
proteins
Identify splice variants:
- do they affect structure?
- do they affect a particular
function?
Predicting receptor three dimensional
structure:
- Sequence lineup of protein
families
- In transmembrane spanning
domains, lipid facing region (‘outer face’) less likely to be conserved
- Highly conserved amino
acids tend to be protein buried sequences which form the ‘internal face’
of the receptor
Is the periodicity of conserved
residues compatible with an a-helix structure?
- Hydropathy analysis
- ~25 amino acids in
alpha helical structure to span lipid bilayer
Molecular methods used to identify
key amino acid residues involved in receptor structure/function
- site-directed mutagenesis
- deletion of receptor
regions
- creation of chimeric
receptor hybrids
Molecular events affecting
receptor function
- alternative splicing
of the gene
- ligand selectivity
- G protein-coupling
- Desensitisation
- Constitutive activity
- Cellular location
- RNA editing - see Lodish
4th ed. pp 437-438 (chapter 11)
- Channel properties
- G protein-coupling
- Use of multiple promoters
- Tissue specific expression
Post-translational modifications
- Amino terminal N-linked
glycosylation
- Important for trafficking
to plasma membrane
- Formation of disulphide
bridges
- Important for three
dimensional structure
- Carboxyl terminal palmitoylation
of a cysteine residue
- Another point of interaction
with the membrane
- Phosphorylation
- Reversible
- Important for receptor
desensitisation and perhaps for allowing receptors to switch to new partners
and signalling pathways
Molecular
Biology of Ionotropic Receptors
Molecular
Biology of G Protein-Coupled Receptors
Further Reading:
- Wess, J. (1997) G-protein-coupled
receptors: molecular mechansisms involved in receptor activation and selectivity
of G-protein recognition. FASEB Journal 11, 346-354.
- Buscher, R, Herrmann,
V and Insel, PA (1999) Human adrenoceptor polymorphisms: evolving recognition
of clinical importance. Trends Pharmacological Science 20, 94-99.
- Ballesteros, JA, Shi,
L & Javitch, JA (2001) Structural mimicry in G protein-coupled receptors:
implications of the high-resolution structure of rhodopsin for structure-function
analysis of rhodopsin-like receptors. Molecular Pharmacology 60, 1-19.
- Seeburg, PH, Higuchi,
and Sprengel, R (1998) RNA editing of brain glutamate receptor channels:
mechanism and physiology. Brain Research Reviews 26, 217-229.
- Moghal, N and Sternberg,
P W (1999) Multiple positive and negative regulators of signalling by the
EGF-receptor. Current Opinion Cell Biology 11, 190-196. (Available at Glasgow
University library)
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