Role of C-Terminal Tails of G Protein Coupled Receptors on Beta-Arrestin 1/2 Dependent Signaling
- Author(s): Pal, Kasturi
- Advisor(s): Defea, Kathryn A
- et al.
Protease activated receptor 2 (PAR2) and Neurokinin 1 receptor (NK1R) are 7 transmembrane receptors (7TMRs), which signal by G alpha q leading to Ca2+ release and protein kinase C (PKC) activation. Both receptors are desensitized by beta-arrestin binding to their C termini. They can also activate ERK1/2 through beta-arrestin scaffolding complexes. They differ in ERK1/2 mediated physiological outcomes: cell migration versus proliferation. Using beta-arrestins, PAR2 activates cofilin, to promote chemotaxis, which was not observed in NK1R. We hypothesized that the differences in beta-arrestin dependent signaling by these 7TMRs can be attributed to how the molecular scaffolds bind the C-tail of the receptors. Using wild type and C-tail chimeras, we showed that the rate of desensitization, internalization, subcellular localization post endocytosis as well as ERK1/2 dependent physiological responses depend on the nature of interaction of beta-arrestins with the C-termini. Bioluminescence resonance energy transfer (BRET) assays showed that PAR2 recruits both beta;-arrestin 1/2 faster and with greater affinity than NK1R. We further show that initial G alpha q signaling is necessary for beta-arrestin 1/2 recruitment to NK1R. PAR2 recruits beta-arrestin 1/2 even when G alpha q pathway is blocked. Assays with C terminal phosphorylation mutants of PAR2 indicated that, phosphorylation of certain residues are necessary for beta-arrestin 1/2 recruitment. Phosphorylation at putative PKC sites (S363 and T366) determine stability of PAR2-beta-arrestin 1/2 complex. This in turn ensures downstream cofilin activation and cell migration. BRET assays with PAR2 and G-protein coupled receptor kinase-2 (GRK-2), revealed that GRK2 is recruited to PAR2 in a dose dependent fashion. It is possible that GRK-2 maybe another kinase which regulates PAR2 signaling. Finally, using allergic proteases from Alternaria alternata and Blatella germanica we demonstrated that PAR2 activation by these proteases leads to beta-arrestin 1/2 recruitment and subsequent beta-arrestin 1 dependent cofilin activation and cell migration. Cell migration brought about by the beta-arrestin signaling arm of PAR2 might be an important molecular mechanism for migration of immune cells to the airways, which is an important symptom of allergic asthma.