The human complement component, C5a, binds two different seven-transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G-protein-coupled receptor that couples to the G␣ i subfamily of heterotrimeric G-proteins and -arrestins (arrs) following C5a stimulation. Peptide fragments derived from the C terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5a pep , in terms of G-protein coupling, arr recruitment, endocytosis, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation at the human C5aR1. We discover that C5a pep acts as a full agonist for G␣ i coupling as measured by cAMP response and extracellular signal-regulated kinase 1/2 phosphorylation, but it displays partial agonism for arr recruitment and receptor endocytosis. Interestingly, C5a pep exhibits full-agonist efficacy with respect to inhibiting lipopolysaccharide-induced interleukin-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared with C5a, although both these responses are sensitive to pertussis toxin treatment. Taken together, our data reveal that compared with C5a, C5a pep exerts partial efficacy for arr recruitment, receptor trafficking, and neutrophil migration. Our findings therefore uncover functional bias at C5aR1 and also provide a framework that can potentially be extended to chemokine receptors, which also typically interact with chemokines through a biphasic mechanism. cro ARTICLE
β-arrestins (βarrs) critically mediate desensitization, endocytosis and signaling of G Protein-Coupled Receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to selectively modulate GPCR functions have not been fully explored yet. Here, we have identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.
SummaryDesensitization, signaling and trafficking of G protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, β-arrestins (βarrs). The two isoforms of βarrs (βarr1 and 2) share a high degree of sequence and structural similarity, still however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of βarr isoforms is still lacking. Using a set of complementary approaches including antibody fragment based conformational sensors, we discover structural differences between βarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain swapped chimeras of βarrs display robust complementation in functional assays thereby, linking the structural differences between the receptor-bound βarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of βarr isoforms to drive distinct functional outcomes, and underscore the importance of integrating this aspect in the current framework of biased agonism.
Highlights d b-arrestin 1 and 2 differentially regulate signaling and trafficking of GPCRs d There are conformational differences between receptorbound b-arrestin 1 and 2 d Domain-swapped chimera of b-arrestin 1 and 2 exhibit functional complementation d Functional differences of b-arrestin isoforms have implications for biased agonism
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