| 1. Objective:G proteins, short for guanine nucleotide-binding proteins, are a family of membrane proteins involved in second messenger cascades. Receptor-activated G proteins are bound to the inside surface of the cell membrane, which is coupled winth its receptos penetrating the membrane 7 times. The trimers G proteins are made up of alpha (α), beta (β), and gamma (γ) subunits. G proteinβγsubunits are dimers, which were no function before. Recently G proteinβγsubunits were found that they are the same as Gαto take part in signal transduction in the cells. The newly reports found that G proteinβγsubunits are not only for signal transduction but also a cross talking point of regulation in signal transductio.We designed the experiments of effect of G proteinβγsubunits on phosphorylation mediated by G-protein coupled receptor kinase 2 (GRK2) of G-protein coupled receptors such as m2 muscarinic acetylcholine receptor (m2 receptor) andβ2-adrenergic receptor (β2-AR) by using Gβγ, m2 receptor,β2-AR and GRK2. Does G proteinβγsubunits could directly activate the activity of some target moleculars such as G-protein coupled receptor kinase 2 (GRK2)? We studied the possible effects of G proteinβγsubunits on GRK2-mediated phosphorylation of G-protein coupled receptors such as m2 muscarinic acetylcholine receptor (m2 receptor) andβ2-adrenergic receptor (β2-AR), To evaluate the role of Gβγsubunits in further modulation of G-protein coupled receptors such as m2 muscarinic acetylcholine receptor (m2 receptor) andβ2-adrenergic receptor (β2-AR). How Gβγsubunits affect m2 receptor andβ2-AR phosphorylation mediated by G-protein coupled receptor kinase 2 (GRK2), to evalute that the Gβγsubunits may regulate the activity of GRK2 in the route of G protein signal transduction and its regulted molecule mechanism.2. Method:1 To purify m2 receptor AChR of rat brain by affinity column chromatography; 2 To purify G protein from porcine brain by using DEAE-Sepharose FF,Sephacryl S-300 HR and Phenyl-Sepharose CL-4B. Finally purify Gβγsubunits from G protein by using GTP and Phenyl-Sepharose CL-4B.3 Purified m2 receptor of rat brain was incubated with GRK2, and [32P] ATP. Proteins were separated by electrophoresis, gels were dried and autoradiograms developed.4 Purified m2 receptor of rat brain was incubated with carbachol, atropine and heparin along with GRK2, and [32P]ATP Proteins were separated by electrophoresis, gels were dried and and autoradiograms developed. And also bands containing m2 receptor were excised and counted by liquid scintillation.5 Purified m2 receptor of rat brain was incubated with purified Gβγsubunits along with GRK2, and [32P]ATP. Proteins were separated by electrophoresis, gels were dried and and autoradiograms developed. And also bands containing m2 receptor were excised and counted by liquid scintillation.6 Purifiedβ2-AR receptor membrane protein was incubated with GRK2, and [32P] ATP. Proteins were separated by electrophoresis, gels were dried and autoradiograms developed.7 Purifiedβ2-AR receptor membrane protein was incubated with turbutaline and heparin along with GRK2 and [32P]ATP. Proteins were separated by electrophoresis, gels were dried bands containingβ2-AR receptor membrane protein receptor were excised and counted by liquid scintillation.8β2-AR receptor membrane protein was incubated with purified Gβγsubunits along with GRK2, and [32P]ATP. Proteins were separated by electrophoresis, gels were dried and autoradiograms developed.3. Result:1. Purified m2 receptors from rat brain by using ABT-agarose affinity chromatography was detected as one primary band with a molecular weight of 68,000 by Coomassie Blue Staining.2 Purified G protein from porcine brain by using DEAE-Sepharose FF,Sephacryl S-300 HR and Phenyl-Sepharose CL-4B. Finally purified Gβγsubunits from G protein by using GTP and Phenyl-Sepharose CL-4B. Purified porcine brain Gβγsubunits as two bands with molecular weight of 36 000 and 10 000, respectively, by Coomassie Blue staining.3. The agonist carbachol evidently increased the phosphorylation of m2 receptor, both atropine and heparin completely blocked m2 receptor phosphorylation by using SDS-PAGE, autoradiograms developed and liquid scintillation.4. The purified Gβγsubunits evidently increased the phosphorylation of m2 receptor by using SDS-PAGE, autoradiograms developed and liquid scintillation.5. The agonist turbutaline increased the phosphorylation ofβ2-adrenergic receptor, the heparin completely blocked m2 receptor phosphorylation by using SDS-PAGE, autoradiograms developed and liquid scintillation.6. The purified Gβγsubunits increased the phosphorylation ofβ2-adrenergic receptor by using SDS-PAGE, autoradiograms developed and liquid scintillation.4. Discussion:The m2 receptors andβ2-AR are coupled to G proteins and regulation of these types of receptors is initiated by phosphorylation by one of a family of protein kinases referred to G protein coupled receptor kinases (GRKs). GRK2, involved in m2 receptor andβ2-AR regulation, was originally termedβ-adrenergic receptor kinase orβARK due to earlier recognition of its role inβ2 adrenergic receptor regulation. GRK2 is now known to phosphorylate other G protein-coupled receptors including muscarinic m2 receptors andβ2-AR. Currently studies have shown the regulation of agonist, antagonist and other chemicals, such as carbachol, atropine, mastoparan or related peptides andβ-arrestin-1, on the agonist-dependent phosphorylation of m2 mAChR.Muscarinic m2 receptors andβ2-AR are regulated by three processes: desensitization, internalization and down-regulation which occurs following receptor phosphorylation by GRK2. Phosphorylation of residues in the third intracellular loop by GRK2 has been shown to be essential for agonist-induced desensitization. In fact, downregulation of postsynaptic receptor density is considered a prominent mechanism for development of tolerance to persistent AChE inhibition. Furthermore, a number of studies have reported direct interaction between muscarinic receptors and a number of toxicants. These direct interactions with muscarinic receptors, with the M2 subtype generally being more sensitive, may provide a mechanism for selective toxicity with different toxicants. Muscarinic receptors andβ2-AR are coupled to G-proteins, and their regulation is mediated by a G protein coupled receptor kinase (GRK). Regulation of both muscarinic m2 receptors and B2-adrenergic receptors is mediated specifically by the isoform GRK2. Phosphorylation of m2 receptor is generally assumed to be involved in the desensitization and in particular, the agonist-dependent phosphorylation of G protein linked receptor is thought to take part in agonist-specific or homologous desensitization. Desensitization plays an important role in turning off receptor-mediated signal transduction pathways. In GPRs, two processes associated with the rapid phase of desensitization are receptor/ G protein uncoupling and receptor internalization. Agonist-stimulated activation of receptor phosphorylation by GRK2 appears requisite for their desensitization, internalization and downregulation. Thus, we hypothesized that some OP toxicants may selectively bind to M2 receptors andβ2-AR and affect GRK2 pathways mediating receptor adaptation. The third intracellular loop of the m2 muscarinic receptor has been shown to be essential for agonist induced desensitization. Agonist-induced phosphorylation of the m2 receptor occurs at two clusters of serine and threonine residues (amino acids 286-290 and 307-311) in the third intracellular domain. These clusters of serine/threonine residues are homologous to region in the a2-adrenergic receptor shown to undergo agonist-mediated phosphorylation. Phosphorylation of residurs 307-311 and coupling of b-arrestins appears important for receptor internalization. As these processes are differentially coupled to m2/m4 (inhibition of adenylyl cyclase) and m1/m3 (phosphoinositide specific phospholipase C activation) muscarinic receptor subtypes, it appeared that the agonist could selectively modify m2 and /or m4 muscarinic receptor function. Other studies suggest that the agonist may affect multiple components of the cAMP cascade system, for instance, their activity and expression of adenylyl cyclase.The agonist carbachol and turbutaline evidently increased the phosphorylation of m2 receptor andβ2-AR respectively, both atropine and heparin completely blocked m2 receptor orβ2-AR phosphorylation, To evaluate further modulation selectively of m2 receptor andβ2-AR phosphorylation by carbachol and turbutaline, phosphorylation ofβ2-AR was carried out as with m2 receptor and phosphorylation of m2 receptor with turbutaline by GRK2. The effects of carbachol, turbutaline and heparin on the phosphorylation ofβ2-AR and m2 receptor by GRK2 also were done. The protein bands showed no apparent concentration dependent effects ofβ2-adrenergic receptor phosphorylation by carbacol and of m2 receptor phosphorylation by turbutaline by GRK2. The results suggest that carbachol only affects GRK2-mediated phosphorylation of m2 and has little effect onβ2-AR phosphorylation; turbutaline only affects phosphorylation ofβ2-AR and has little effect on m2 receptor phosphorylation. These results suggest that these agonist and the antagonist may bind directly to the receptors, possibly at the GRK2-sensitive residues in the third intracellular loop. These agonist and antagonist may therefore alter GRK2-mediated regulatory pathways for receptors by direct interaction with the receptors. These differential actions could contribute to selective differences in the regulation of receptors following exposure to the agonist and the antagonist.Purified rat brain M2 receptor was detected as one primary band with a molecular weight of 68,000 and purified porcine brain Gβγsubunits as two bands with molecular weight of 60,000 and 10,000, respectively, by Coomassie Blue staining. Purified Gβγsubunits was incubated with purified M2 receptor, orβ2-AR along with GRK2, and [32P]ATP. The antagonist atropine, the GRK2 inhibitor heparin on the phosphorylation of m2 receptor and agonist turbutaline, heparin onβ2-AR phosphorylation by G-protein coupled receptor kinase 2 (GRK2) were tested. Purified m2 receptor of rat brain andβ2-AR membrane protein were incubated, respectively, with agonist carbachol and turbutaline along with GRK2, and [32P] ATP in the presence of the agonist carbachol and turbutaline.Paired tubes contained the agonist and antagonist or the GRK inhibitor heparin. Proteins were separated by electrophoresis, gels were dried and autoradiograms developed. Bands containing receptors were excised and counted by liquid scintillation. The G proteinβγsubunits evidently increased the phosphorylation of m2 receptor without carbachol andβ2-AR without turbutaline, respectively, mediated by GRK2. The agonist carbachol and turbutaline evidently increased the phosphorylation of m2 receptor andβ2-AR respectively, both atropine and heparin completely blocked m2 receptor orβ2-AR phosphorylation. The protein bands showed no apparent concentration dependent effects ofβ2-adrenergic receptor phosphorylation by carbacol and of m2 receptor phosphorylation by turbutaline by GRK2. The results suggest that carbachol only affects GRK2-mediated phosphorylation of m2 and has little effect onβ2-AR phosphorylation; turbutaline only affects phosphorylation ofβ2-AR and has little effect on m2 receptor phosphorylation. These results indicated that G proteinβγsubunits could regulates the activity of GRK2. It may increases the the activity of GRK2. It told us that G proteinβγsubunits has an important effects on some protein moleculars, the same as Gαsubunit. G proteinβγsubunits has an important role in the signal transduction of the cells.5. Conclusion:1. The purified Gβγsubunits evidently increased the phosphorylation of m2 receptor by using SDS-PAGE, autoradiograms developed and liquid scintillation.2. The purified Gβγsubunits increased the phosphorylation ofβ2-adrenergic receptor by using SDS-PAGE, autoradiograms developed and liquid scintillation.3. The rrsults indicates that the purified Gβγsubunits increases the activity of GRK2.
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