Roles of proteolysis in regulation of GPCR function

Abstract

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects on biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors, or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating GPCRs. At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevent signalling. Conversely, cell-surface peptidases can also generate bioactive peptides, which directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signalling. Certain peptidases can signal directly to cells, by cleaving GPCR to initiate intracellular signalling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signalling and mediate down-regulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signalling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signalling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signalling in disease.

Figure 1

Cell-surface peptidases regulate GPCR-mediated signalling. (A) ACE compound peptidase cleaves angiotensin I to generate angiotensin II to promote activation of angiotensin II receptors. Conversely, NEP hydrolyses SP to prevent activation of SP of neurokinin 1 receptors. (B) Activation of certain GPCRs promotes the ADAMs that generate EGF-like ligands (e.g. heparin-binding EGF-like factor, amphiregulin). In turn, these ligands transactivate ErbB receptors to activate intracellular signalling pathways.

Figure 2

Peptidases act as biased agonists at GPCRs. (A) Trypsin cleaves PAR₂ to create a new N-terminus that activates PAR₂ mobilizing intracellular calcium and promoting phosphorylation (p) of ERK1/2. (B) Cathepsin G cleaves PAR₂, but does not elicit any known signalling and prevents activation by other peptidases such as trypsin. (C) Other peptidases such as elastase-2, cleave PAR₂ at a site distinct from trypsin. The action of this peptidase does not mobilize intracellular calcium, but does activate ERK1/2.

Figure 3

USPs promote lysosomal degradation and recycling of GPCRs. (A) (1) USPs cleave ubiquitin molecules from GPCRs to (2) promote entry in lysosomes and degradation by lysosomal peptidases or (3) recycling of GPCRs to the cell-surface mediating resensitization of signalling. (B) Ubiquitination of β-arrestins promotes GPCR-mediated phosphorylation of extracellular-regulated PK 1 and 2. (2) De-ubiquitination of β-arrestins by USPs destabilizes the GPCR•β-arrestin complex to terminate ERK1/2 activation.

Figure 4

Endosomal peptidases promote GPCR recycling and resensitization. (1) Vacuolar-type H⁺-ATPases pump protons (H⁺) into vesicles, acidifying early endosomes. (2) Peptide agonists such as SP and CGRP have reduced affinity for their respective GPCRs. SP and CGRP become substrates for the endosomal peptidase, ECE-1 at low pH and are hydrolysed to inactive metabolites. (3) β-Arrestins dissociate from the GPCR, returning to the cytosol. (4) The GPCR, free from β-arrestins then recycles back to the cell-surface to mediate resensitization. (5) Certain GPCRs (e.g. neurokinin-1 receptor) signal from endosomes in a β-arrestin-dependent mechanism, phosphorylating extracellular-regulated PKs 1 and 2 (pERK1/2). ECE-1 promoted dissociation of β-arrestins terminates ERK1/2 activation. (6) β-Arrestins can recruit protein phosphatases such as protein phosphatase 2A (PP2A) to desensitized GPCRs at the cell-surface. (7) PP2A activity dephosphorylates cell-surface located GPCRs promoting resensitization.