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Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with β-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a β-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.

In this issue of the Biochemical Journal, Xu et al. describe how they use a spot peptide array to identify a unique sequence within beta-arrestin-2 that is required for both multimerization and ERK1/2 (extracellular-signal-related kinase 1/2) scaffolding. They provide evidence that dimers may serve as more than just 'storage forms' of beta-arrestins, incapable of interacting with receptors but, rather, perhaps, adding to the specificity of G-protein-coupled-receptor signalling. They show that key charged residues within this dimerization interface of beta-arrestin-2 block association with ERK1/2 and subsequent activation of ERK1/2 by beta(2)-adrenergic receptors, while internalization is unaffected. They suggest that self-association may serve as a means of sheltering scaffolding sites on beta-arrestins from specific binding partners to prevent constitutive activation of key signalling pathways. These studies enhance our understanding of how beta-arrestins can juggle their roles as scaffolds of multiple pathways in response to diverse signals.

PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.