BS3054:TOPIC FOUR

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

Rhodopsin

- pigment in rod cells that sense light

- organised in rows in intercalating discs

- N terminal is in intercalating discs and C terminal is in cytoplasm

- when activated by photon of light = causes G-protein DEPENDENT signalling = NT release from cell

rhodopsin regulation by GRK1

- Rhodopsin kinase (GRK1) is on the membrane = sequentially phosphorylates c terminal of active rhodopsin

- rhodopsin recruits arrestin 1 = has high affinity for phosphorylated serine

- GPCR-arrestin complex recruits AP2 and clathrin = endocytosis

- allows translocation to a point where the receptor can be endocytosed

Life cycle of a GPCR in rod cells

1. desensitisation = active GPCR is phosphorylated by GRKs

- recruits arrestin

- recruits AP2 and clathrin

- translocates to mebrane for endocytosis = forms clathrin coated vesicle

- recruits dynamin to separate from membrane and complete endocytosis

2. Sequestration

- endosome reduces its intracellular pH = acidification of the vesicle

- causes dissociation of the ligand and disassembly of the receptor-arrestin complex

3. phosphatases dephosphorylate the receptor = recycled or degraded

4. resensitisation of receptor or synthesis or new receptors

GRK isoform groups

GRK1 = rod rhodopsin kinase (VISUAL)

GRK7 = cone rhodopsin kinase (VISUAL)

GRK2 = B-ARK1 (NON-VISUAL)

GRK3 = B-ARK2 (NON-VISUAL)

GRK4

GRK5 } = NON- VISUAL

GRK6

Characteristics of GRK2 and GRK3

- Catalytic domain

- RH domain = RGS-like homology domain (binds Gaq)

- PH domain = binds phospholipids

- beta gamma domain = binds Gbeta-gamma

= domains that allow Gaq an Gbg association as well as PL association = allows scaffolding to the membrane

Characteristics of GRK4, GRK5 and GRK6

- Have PIP2 binding domain (phospholipid association)

- Have fairy homologous catalytic domain to GRK2 and 3

- RH? domain = looks like RH domain but no evidence that is allows Ga binding = potentially lost its function?

- GRK4 and 6 contain lipid modifications = permanently associated with the plasma membrane

- GRK5 has polybasic region = associated with membrane

Histological imaging of GRK2 vs GRK6 shows what?

- GRK2 is localised to the cytoplasm

- GRK6 = predominantly associated with the membrane

GRK2/3 recruitment to receptor

- via G-beta-gamma

- via G-alpha due to RH domain

GRK2 interactome

- research being done into what else GRKs do = interactome

- shown to have involvement with PKC, PKA, Ca2+ calmodulin signalling etc.

GRK-based therapeutics

- b2 adrenoceptor agonists for treatment of obesity and diabetes II

- selective for GRK2 - no Gas or arrestin recruitment otherwise would cause side effects in heart or desensitisation of receptors

- they enhance muscle metabolism and metabolic health

Why does the body not require many forms of types of arrestin

- highly flexible so can fold into many different conformations to bind many different receptor types

types of arrestin

- arrestin 1 (visual) - rod

- arrestin 4 (visual) - cone

- arrestin 2 (non-visual)

- arrestin 3 (non-visual)

Can GPCRs signal without G-proteins

- evidence in slime mould that they can - deleted beta subunit so G-protein dependent activation couldn't occur

- increasing cAMP levels stimulated cAMP GPCRs

Can arrestins scaffold proteins other than AP2 and clathrin

- been shown that the arrestin complex can recruit Src (non-receptor tyrosine kinase) = activates MAPK signalling via ERK scaffold

= ERK scaffold - Raf, ERK and MEK (MAPK)

Signalling pathways of dependent vs independent G-protein signalling

- G-protein dependent

= adenylyl cyclase, PLC, PI3K etc

- G-protein independent

= c-Src, MAPK, DAG kinase etc.

biased ligands

- biased ligands can distinguish between modes of GPCR signalling = allows selective transduction via dependent/ independent pathways

selective recruitment of arrestins

- cells that are acted on by the same GRK can recruit different arrestins

- e.g. endothelin-1 receptor recruits arrestin 3 and UTP receptor recruits arrestin 2

- both cell types are in the blood vessels but have differing roles

what might determine cell-specific arrestin signalling

- may be determined by phosphorylation signature on the receptor

- conformation of arrestin might determine what the protein scaffolds with