BIOC 222 - LECTURE 27

how many transmembrane domains does a GPCR have

7

which terminal is extracellular

N

whcih terminal is intracellular

C

what are the classes of ligands and what are they classified based on

• agonist

• inverse agonist

• antagonist

• classified based on whether they occupy the receptor, or alter the activation state

describe the confromational state that receptors sit in when unbound

• sit in a conformational equilibrium between the on state and the off state

describe what an agonst does to the receptor

• stabilise the on state

• activate the receptor

describe what an inverse agonist does to the receptor

• stabilises the off state

• deactivates the receptor

describe what an antagonist does to the receptor

• equillibrium unchanged

• neither activates or deactivates the receptor

what type of ligands are endogenous ligands usually

• agonists

what site do endogenous agonists bind to

orthosteric sites

how many drugs can bind to an orthosteric site at one time

1

what do orthosteric agonists mimic

activity of the endogenous ligand activity

although may be more or less efficacious

what are the subunits of a G protein

• a

• b

• y

describe the association between beta and gamma

they are obligatory dimers

they are synthesised together and never seperate

describe the G protein in its resting state

• alpha sub unit has GDP bound

• all three units are associated

describe what happens when receptor gets activated

• it interacts with the G protein

• it promotes GDP release from alpha sub unit

• GTP binds alpha

• alpha and beta gamma have downstream effects

• alpha sub unit hydrolyses GTP to GDP which terminates its signal

what does alpha sub unit do

activate effector proteins such as adenylate cyclase

• AC increases secondary messenger cAMP which has downstream effects

describe what happens to the activated receptor - that prevents further G protein coupling

• recruits G protein receptor kinases

• they phosphorylate intracellular domain which promotes arrestin binding

• arrestin prevents further G protein coupling > desensitisation

describe how the receptor is internalised

• arrestin and clathrin and other scaffolding molecules are able to internalise the receptor

• it can then either be recycled back into the membrane or degraded

describe the conformational change of the receptor when an agonist binds

• 2 salt bridges in CB1 are broken

• leads to outward movement of transmembrane 6 and 2

• this opens up the lower part of the receptor which enables G protein to interact

what is rhodopsin

GPCR for light

describe how rhodopsin works

• it has retinal, which is its ligand, bound in the inactive state

• when light is present, retinal isomerases into a different conformation, which activates the receptor

describe the basal activity of rhodopsin compared to other receptors

• it has super low/no basal activity and is locked and loaded in the inactive state

• other receptors have some basal activity

why does rhodopsin not have basal activity

• rhodopsin has a strongly favoured inactive state with a large energy barrier between the active and the inactive state

• therefore it cannot readily switch between the two in the absence of a ligand

• therefore it remains in the inactive state

• ligand comes along and stabilises the active state

describe what gives other receptors basal activity

• whereas other receptors have lower energy differences between the active and inactive state and therefore sample the active state in the absence of a ligand which produces basal activity

• ligands stabilise the active state

Does the agonist alone produce one fixed active shape?

• no when the agonist binds the receptor samples altered or intermediate conformations.

• when the G protein binds, it stabilises the active conformation

• therefore structures obtained with the G protein bound and without may be different

what are partial agonists

they cannot fully activate the receptor even at 100% occupancy

what are the theories for how a partial agonist produces an incomplete activation

equillibrium theory

unique active state theory

describe the equillibrium theory

• stabilise a different equillibrium between active states

• bind to a receptor and sometimes activate it and sometimes inactivate it and so the overall efffect is just a partial agonism

evidence behind equillibrium theory

B2AR

• three predominant conformations, including differences around the ligand-binding region and the movements in the TM that occur upon binding

• different ligands differ in how many receptors the stabilise in each conformation

• so partial agonism stabilises a proportion of these states that doesnt provide a full activation

• whereas agonism stabilises a different proportion of these states that does provide a full response

describe the different active states theory

A2A

• 2 inactive states

• 2 active states

• partial agonist stabilises one active state

• agonist stabilises the other active state

describe B arrestin biased ligand effect

• forces the transmembrane helices of the receptor into a specific shape that does not couple to G proteins.

• Instead, it exclusively promotes phosphorylation of the receptor tail and recruits -arrestin

• B arrestin desensitises G protein signalling and pulls the receptor inside the cell

describe G protein biased ligand effect

•  it stabilizes a distinct receptor conformation that recruits and activates G proteins

• the receptor isn't phosphorylated, -arrestin is never recruited

describe the effect of opiods and what mediates them

• pain relief but also respiratory depression and constipation and tolerance

• theory is that G protein pathway mediates pain relief and the B arrestin pathway mediates respiratory depression, constipation and toelrance

what can biased ligands be used for in terms of improving opiods

• activate only the G protein pathway

• reduce side effects from B arrestin pathway