G PROTEIN COUPLED RECEPTORS - AI generated

Structure and function of GPCRs

G-Protein coupled receptors are the largest family of cell surface receptors

-        Sight smell and taste depend on them

-        >800 in humans

-        Characterised by seven membrane spanning alpha-helical segments separated by alternating intra and extracellular loop regions forming a cylindrical structure with a deep ligand binding at the centre

-        Use G proteins to relay signal into the cell

-        Highly conserved

G Proteins

Ligand binding causes confirmation change which activates the G protein to transduce signal into the cell.

-        G proteins are GTP binding proteins

-        Have alpha beta and gamma subunits

Alpha subunit binds to either GDP or GTP

-        When active bound to GTP

-        When inactive bound to GDP

Have intrinsic GTPase activity

-        GTPase activating proteins (GAPs) inactivate the GTP-binding protein by stimulating the hydrolysis of GTP to GDP

-        Guanine nucleotide exchange factors (GEFs) activate the G proteins by stimulating it to release GDP allowing GTP to bind.

There are two types, monomeric and trimeric

-        In monomeric the GTP binds directly to the protein

-        In trimeric the GTP binds to the alpha subunit of the protein and the activated protein serves as the GEF

Activated G proteins continue the signal through second messengers

-        Cyclic nucleotides, phospholipids and calcium ions

-        These phosphorylate targets leading to changes in the cell

Desensitisation

GPCR desensitisation depends on receptor phosphorylation

When target cells are exposed to a high concentration of a stimulating ligand for a long time the become desensitised or adapted

-        Receptor sequestration, receptors are temporarily internalised so they no longer have access to their ligand

-        Receptor down-regulation, the receptor is internalised and destroyed by lysosomes

-        Receptor inactivation, can no longer interact with G proteins

The desensitising depends on the phosphorylation by PKA, PKC or a number of GPCR kinases (GRKs)

-        GPCR is activated and stimulates GRKs to phosphorylate multiple serine’s and threonine’s which causes arrestin to bind which means it cannot interact with G proteins

-        It causes the receptor to be coupled with clathrin-dependent endocytosis machinery inducing GPCR endocytosis, there are three outcomes

o   GPCR is dephosphorylated and recycled

o   Ubiquitated and degraded in lysosomes

o   Internalised complex recruits other signalling proteins activating new pathways but this is a diff signalling pathway as it does not use G proteins

Complexity of GPCR Signalling

The linear pathway would be

Adrenaline → β2-adrenergic receptor (β2AR) → activation of stimulatory subunit of heterotrimeric G protein (Gαs) → stimulation of adenylyl cyclase → accumulation of cAMP → activation of cAMP dependent protein kinase A (PKA) →phosphorylation of proteins to transduce signal

But this is not the case as GPCRs are not switches

-        Β2AR exhibits significant constitutive activity which can be blocked by inverse agonists.

o   Β2AR couples to both Gαs (when bound to agonist) and the inhibitory subunit Gαi (when bound to inverse agonist

o   Β2AR can signal through MAP kinase pathways in a G protein independent manner through arrestin

-        GPCR desensitisation involved multiple pathways, receptor phosphorylation, arrestin-mediated internalisation into endosomes, receptor recycling, lysosomal degradation

-        GPCR oligomerisation and dimerization

-        Localisation to specific membrane compartment with differences in lipid-bilayer composition

Multifaceted functional behaviour has been observed for many difference GPCRs, ligands can be grouped into difference efficiency classes

-        Full agonists, maximal receptor stimulation

-        Partial agonists, unable to elect full activity even at saturating concentration

-        Neutral agonists, no effect on signalling activity but can prevent other ligands from binding. Also called antagonist

-        Inverse agonists, reduce the level of basal constitutive activity below that of unliganated receptors

The different ligands stabilise distinct GPCR conformational states leading to difference activated

-        A specific ligand binds which engages with distinct subsets of conformational switches which stabilises a distinct conformational state so the GPCR interacts with specific effectors activating the specific signalling pathway

Importance of GCPR Signalling

GCPRs are the largest family of membrane proteins and are activated by a spectrum of ligands including hormones, neurotransmitters, ions, photons and others. So they play a role in an array of functions in the human body

-        Half of marketed drugs work on GCPRs

GCPR role in olfaction

A subset of GCPRs are called olfactory receptors (ORs) olfaction is the sense of smell

-        700 in humans with only half functional

-        1,200 in rodents with 2/3 functional as rodents have a much more sensitive sense of smell

The large OR family evolved to detect large numbers of diff chemical structures in the environment

-        Animals can detect and discriminate thousands of low molecular mass organic odours

-        Both aliphatic and aromatic compounds with diff carbon backbones and aromatic compounds

ORs have extensive sequence diversity within the transmembrane domain which is the site of ligand binding to detect the variety of chemical structures

Structure

At the back of the nose there is an olfactory epithelium which contains olfactory sensory neurones which have cilia which extend into the nasal cavity and have the ORs. The axons extend into the brain to the area smells are detected call olfactory bulbs

Process

1.      OR binds ligand

2.      Couples with G protein and Gα_olf (a Gαs isoform enriched with OSNs)

3.      Gα_olf activates adenylyl cyclase which increases cAMP

4.      Increase in cAMP opens cyclic nucleotide gated channels (CNG channels) which increases Na+ and Ca^2+ influx

5.      Depolarisation of neurone       

6.      Initial depolarisation amplified by subsequent activation of Ca^2+ activated Cl- channels and efflux of Cl- from the cell

7.      Odour induced depolarisation in olfactory cilia spreads throughout the neurone

8.      Results in opening of voltage sensitive ion channels and the firing of action potentials

9.      Release of neurotransmitter at the synaptic terminal in olfactory bulb

Identification

To determine the identity of the odorant stimulus the nervous system must discern which of the 1000s of OR have been activated

There are three organising principles

1.      Each OSN in the olfactory epithelium expresses a single allele of a single OR – one receptor one neurone

2.      OSNs expressing the same ORs project to a common glomerulus in the olfactory bulb

3.      A single receptor can recognise multiple odorants and a single odorant is recognised by multiple receptors

a.      The nose uses combinational coding to discriminate between a fast number of different smells so the olfactory system can uncoded an unlimited number if odorants

b.      Individual odorants can activate diff subset and numbers of ORs and with different magnitudes which allows them to discriminate between different odorants with similar structures are they are recognised by different but overlapping sets of receptors

c.      OR dependant pattern of connects and intrinsic activity of OR sets the level of intracellular cAMP which in turn regulates the expression of the axon guidance receptor Neuropilin 2

                                                                                             i.     Increase in CAMP causes posterior shift in innervation

                                                                                           ii.     Decrease in cAMP causes anterior shift in innervation

d.      ORs are important in the convergence of OSNs expressing the same OR