Phosphorylation-independent Desensitization Of MGluR5 Mediated By GRK2

Metabotropic glutamate receptors (mGluRs) serve important neuromodulatory roles at glutamatergic synapses to shape excitatory neurotransmission. The desensitization of mGluRs is an important mechanism in regulating the functions of the receptors. The G protein-coupled receptor kinases (GRKs) have been shown to regulate the desensitization of many GPCRs. The desensitization of mGluR1 is in response to both GRKs and second-messenger-dependent protein kinases. Moreover, the desensitization of mGluR1 mediated by GRK2 appears to be both phosphorylation- andβ-arrestin-independent. These studies identify novel roles for GRK2 in regulating mGluR5 that may serve to further shape the function of these receptors in neurotransmission.In this study, we research the signaling of mGluR5 mediated by GRK2. Using molecular cloning technology, we constructed a catalytically inactive GRK2 mutant (GRK2-K220R) and a mutant of mGluR5 (mGluR5-del847-T840A) which lack of potential phosphorylation sites. Then using ELISA detected the expression of mGluR5 and mGluR5-del847-T840A in the absence or presence of GRK2/GRK2-K220R, and then using IP3 formation detected the effects of these mutants on the regulation of mGluR5. We found that the desensitization of mGluR5 mediated by GRK2 is phosphorylation -independent, and the changes of the receptors expression in membrane were not contributed to the desensitization. At the same time, we constructed the whole length of GRK2 and five splice mutants containing different domains of GRK2 which are all labelled with HA epitope. Then the expression of GRK2 mutants was assessed by ELISA and Western Blot. The results demonstrated that the GRK2 and the five splice mutants could express in HEK293 cells. The results will provide a good basis for exploring which domain of the GRK2 participating in the receptor desensitization.In a brief, our study has confirmed that the desensitization of mGluR5 mediated by GRK2 is phosphorylation-independent, and the changes of receptors expression in membrane were not contributed to the desensitization. Our results will provide a good foundation for exploring the clear mechanism of the desensitization of GPCRs mediated by GRK2.