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the likely substrates of the very large number of protein kinases involved in cell

signalling and to study the role of ubiquitination in receptor recycling.

• The use of theenzyme fragment complementation(EFC) technique. This is commonly
  carried out using the enzymeb-galactosidase. The technique uses a small fragment of
  the enzyme and an inactive deletion mutant of the enzyme both of which are
  enzymically inactive. The two forms are attached either by chemical conjugation or by
  a recombination procedure to two proteins suspected of interacting in a particular
  signalling pathway. If the two proteins do interact they allow the two attached
  galactosidase forms to complement each other generating a heteromeric complex

(i) Receptor binding

Radioligand filtration

Radiolabeled SPA

FP

(ii) G protein
binding

(iii) Second messengers

cAMP

Ca2+

IP 3

(iv) Protein redistribution

b–Arrestin translocation

Receptor internalization

Plasma

membrane

b–arrb–arrestin

translocation

Ca2+

IP 3

cAMP

Ga C-terminal

peptide

analogs

(ii) G protein activation
GTPgS

A = Agonist

A A A

PLC AC

Nuclear

membrane

Transcription

GPCR

internalization

Reporter genes

Luciferase

b–lactamase

b b b

ai g g aq g

RRR

R

as

Fig. 17.6.Examples of GPCR screening approaches. The modulation of GPCR activity can be measured using a

variety of assays, including (i) receptor binding; (ii) G-protein binding and activation; (iii) second-messenger

signalling, including reporter systems that measure the response downstream; and (iv) protein redistribution.

(i) Receptor binding can be assayed using filtration binding of a radiolabelled ligand using a radioactive ligand

and a scintillation proximity assay (SPA), or using a fluorescently labelled ligand and fluorescence polarisation

(FP) to monitor specific binding between the ligand and the receptor. Receptor activity can also be monitored

using (ii) G-protein binding and activation with high-affinity peptides that mimic the GaC-terminus or using

GTPgS assays. Subsequently, Gaand Gbggo on to activate their effector molecules, and (iii) second-messenger

signalling pathways can be monitored. For example, the Gaqsubunit can interact with phospholipase

C (PLC), causing enzyme activation and the catalysis of phosphotidylinositol-4,5-bisphosphate to inositol-1,4,

5-trisphosphate (IP 3 ) which can then mobilise Ca^2 þ. Alternatively, the Gassubunit can interact with and

promote the activity of adenylyl cyclase (AC), resulting in the production of cAMP. When assaying for

(iv) protein redistribution, either the translocation ofb-arrestin (b-arr) or the internalisation of the GPCR can be

monitored. (Reproduced from Gilchrist, A. (2007). Modulating G-protein-coupled receptors: from traditional

pharmacology to allosterics.Trends in Pharmacological Sciences, 28 , 431–438, by permission of Elsevier Science.)

687 17.4 Mechanisms of signal transduction