BS3054 L9 - G-Protein Independent Signalling 1

Tachyphylaxis

Reduction in drug responsiveness brought on by repeated dosing over a short time

- caused by ongoing or sequential additions of an agonist

Clincal trial with Metaproterenol

- looked at bronchial epithelial and alveolar cells (contain B-adrenoceptors) before and after stimulation with a B-adrenoceptor agonist (metaproterenol) (6 doses over 24 hours)

- measured adrenocpetor density in the cells and after the stimulation the receptor population had decreased by more than 50% = receptor desensitisation

What are the two types of desentisisation

- homologous = reduction in signalling response is specific to the receptor being stimualted

- heterologous = where 'collateral' receptor signalling is also desensitised i.e. responses affected by receptors not directly exposed to the agonist

How do GPCRs desensitise

- upon exposure to agonist they become covalently modified

= receptor phosphorylation

phosphrylation (densensitisation) of GPCRs

- nearly all GPCRs are rapidly phosphorlyated after agonist stimulation

- occurs at multiple sites: mostly on serine, sometimes on threonine, and rarely on tyrosine residues

- phosphorylation sites are usually in the C-terminal tail and/or third intracellular loop of the GPCR

characteristics of heterologous receptor phosphorylation

- mediated by range of moelcules e.g. G-protein, effector, receptor

- agonist occupied and agonist unoccupied receptors can be phosphorylated

- phosphorylation occurs quite slowly but can occur at low occupancy

what proteins regulate homologous desensitisation

- GRK2-6

- arrestins

How do BARKs work

- B-adrenergic receptor kinase (BARK) also known as GRK2-6

- active receptor activates G-protein which then recruits BARK

- BARK phosphorylates proteins on IC domains and C terminus

Arrestins

- Family of proteins which bind to phosphorylated GPCRs and participate in their desensitization by one of two mechanisms

(1) preventing the receptors from interacting with a G protein, or

(2) serving as scaffolding proteins to couple the receptors to clathrin-dependent endocytosis machinery

- Arrestin sits where the G-protein would sit on the receptor = sterically hinders

GRK and arrestin protein families

Retinal rod and cone cells:

Rod cells = GRK1 + arrestin 1

Cone cells = GRK7 + arrestin4

Non-visual cells:

- GRK2, GRK3, GRK4, GRK5, GRK6

- arrestin 2, arrestin 3

Rhodopsin regulation by GRK1 and arrestin 1

- activated rhodopsin is a substrate for rhodopsin kinase (GRK1)

- multiple phosphorylations of C terminal serine residues of rhodopsin occur = creates high affinity binding site for rod arrestin (arrestin 1)

Clarithrin-mediated receptor endocytosis

- cells internalise specific molecules by forming vesicles on the plasma membrane

- ligand binds to receptor on cell surface = conformational change that allows IC adaptor proteins such as AP2 and clathrin

- adaptor proteins then link to clathrin molcules = assemble and bend the membrane inwards

- calthrin-coated pit continues to invaginate and eventually pinches off = clathrin coated vesicle