The Study Of Coupling Between G_q Protein-coupled Receptor And IP₃ Receptor In Variety Of Cells

The G-protein coupled receptors account for the majority of membrane receptors. There are many kinds of G-proteins, which are composed ofα,β,γsubunits. Theαsubunit has the GTPase binding position which can bind GTP and hydrolyze GTP. According to the structure ofαsubunit, G proteins can be divided into six sub-families: Gs,Gi/o,Gq,Gt,Gg and G12. The PLC signaling pathway leaded by the G-protein coupled receptors is extensively distributed in both neuronal and non-neuronal cells. PLCβ, when stimulated by Gαq proteins, hydrolyze phosphatidylinositol 4, 5-bisphosphate (PIP2), yielding two intracellular second messengers IP3 and DAG, which in turn are involved in varieties of cell functions, such as ion channel endocytosis and exocytosis, membrane attachment to the cytoskeleton,etc.The IP3 receptor is distributed in the endoplasmic reticulum membrane. It is a kind of calcium channel protein which can trigger calcium release from endoplasmic reticulum after binding with IP3. With the rise of calcium in living cells, the calcium signal system will be activated. Consequently, the calmodulin will take its action to modulate many physiological processes. Till now, four kinds of IP3 receptors have been discovered, among which theⅠtype receptor is distributed extensively in the central nerve system. Therefore, the abnormal calcium signal will have an important impact toward many pathological processes.M-current was first discovered in bullfrog sympathetic neurons by Brown and Admas in 1980. M current, namely IM, is a slowly activating, non-inactivating, voltage dependent and potassium current. It is named M current because of its suppression by muscarinic receptor activation. Great efforts have been contributed to elucidate the mechanism of IM inhibition ever since its discovery almost 25 years ago. It has been found that the activation of a majority of Gq protein-coupled receptors will result in IM inhibition, and PIP2 is involved in this inhibitory process. It has been proved that the stimulation of Gq protein-coupled receptor will result in PIP2 hydrolysis, which in turn produces IP3 and DAG. It has been discovered that the M current inhibition made by M receptor activation is attributed to the PIP2 hydrolysis. But the M current inhibition made by BK2 receptor activation is not caused by the hydrolysis of PIP2, but by the rise of intracellular calcium. So here we raise a question: since M1 and BK2 receptor are all Gq protein coupled receptors and they both follow the same PLC signaling pathway, Then why is BK2 receptor capable of rising the intracellular Ca2+ while the M1 receptor is not? Some researchers have proposed a hypothesis that there may be a signaling microdomain that determines the divergence of the same signaling transduction pathway. They showed that BK2 receptor and IP3 receptor are co-localized in a signaling microdomain. Accordingly, the IP3 generated by BK2 receptor activation will gain more opportunities to bind with IP3 receptor, leading to the rise of intracellular Ca2+. By contrast, M1 receptor does not tend to co-localize with IP3 receptor in the same microdomain as BK2 receptor does, Then M1 receptor is distributed more remotely from IP3 receptor. Thus the IP3 generated by M1 receptor activation will diffuse in the cell extensively. In concequence, there will be no enough IP3 binding with IP3 receptor. As a result, the Ca2+ in living cells would not rise. However, the above results have only been tested in rat superior cervical ganglion (SCG). As we all know, the PLC signaling pathway constritutes a virtually universal signal transduction mechanism. So is it possible for these Gq coupled receptors tend to form the same microdomain in other kinds of neurons as well as non-neuronal cells?Previous experiment in our lab shows that the calcium releases made by different Gq protein-coupled receptors differ from each other, and the calcium releases made by the same receptor in different types of neurons tend to show different properties as well. In SCG, the activation of BK2, H1, AT1 receptors can increase the intracellular Ca2+, however, M1 receptor can not. In hippocampus neurons, the activation of M1, BK2, H1 receptors can increase the intracellular Ca2+, but AT1 receptor can not. Besides, the Ca2+ changes in HEK293 cells overexpressing with M1 and BK2 receptors are different from that in SCG. Both of these two receptors are able to raise the intracellular Ca2+ in HEK293 cells Moreover, we overexpressed M1 and BK2 receptors in Xenopus oocytes and the results resemble those of the HEK293 cells. So in the present stud, we use rat SCG and hipocampus neurons, HEK293 cells and Xenopus oocytes to investigate the relationship between different Gq protein-coupled receptors and IP3 receptors in vitro in order to establish biochemistry proof for the diversity of Gq protein-coupled receptor signaling pathway.Objective: By using co-immunoprecipitation technique, to study the coupling of different GPCR (M1,H1,AT1,BK2) with IP3 receptor in neuronal and non-neuronal cells.Methods: Coimmunoprecipitation SDS-PAGE,Western blot are used to investigate the relationship between M1,H1,AT1,BK2 receptors and IP3 receptor in two kinds of neurons, rat SCG and hipocampus neurons, and HEK293 cells and Xenopus oocytes. M1,H1,AT1,BK2 receptors are expressed in HEK 293 cells using liposome transfection kit. M1 and BK2 receptors are expressed in oocytes by means of cRNA microinjection method.Results:⑴The expression of M1,H1,AT1,BK2 receptors in SCG and hippocampus neurons was detected.⑵The expression of IP3 receptor in SCG and hippocampus neurons was detected⑶The coimmunoprecipitation results showed that in SCG, H1,AT1,BK2 receptors could be coimmunoprecipitated with IP3 receptor, however, no coupling was detected between M1 receptor and IP3 receptor. In hippocampus neurons, AT1 receptor could coimmunoprecipitated with IP3 receptor, however, no coupling was detected between M1,H1,BK2 receptors and IP3 receptor. (4) The overexpression of these four kinds of receptors in HEK293 cells was detected. (5)The IP3 receptor in HEK293 cells was also detected. (6)In HEK293 cells, M1, AT1, BK2 receptors could coimmunoprecipitated with IP3 receptor, but H1 receptor could not. (7)The overexpression of M1 and BK2 receptors in oocytes was detected. (8)The IP3 receptor in oocytes was also detected. (9)In oocyte, both M1 and BK2 receptors could coimmunoprecipitated with IP3 receptor.