Molecular Cloning Of Pig Ghrelin Receptor Gene And Pharmacological Characterization Of Its Mutants

Ghrelin receptor (GHSR) is a polypeptide consisted of366amino acids. It belongs to the G protein coupled receptor (GPCR) and it has seven tiers of membrane structures. Ghrelin is a polypeptide contained28amino acids that mainly produce in the stomach. This kind of polypeptide has been certified that it is the endogenous ligand of GHSR. It is researched that GHSR plays a vital role in promoting the secretion of growth hormone (GH), regulating ingestion, energy metabolism, gastrointestinal motility or secretion and some other aspects.Ghrelin receptor also has great influences on regulating the growth of pigs. However, the research on GHSR is restricted to the secretion of GH, gastrointestinal motility, cardiovascular function, cell proliferation. Ghrelin receptor has been found that it has some other biological functions and mechanism of actions from the study on human beings, fishes, birds, but this is still unknown on pigs. Therefore, to know more about ghrelin receptor of pigs, in this study, we successfully establish the eukaryon expression vector of ghrelin receptor of pigs by splicing, overlapping, extending polymerase chain reaction (PCR) to clone the coding sequence of ghrelin receptor of pigs.To know more about pig ghrelin receptor, we selected six mutations:P108L, I134T, V1601, C173R, A204D and F279L, by PCR artificial mutagenesis. Corresponding recombinant receptors were transiently expressed in human embryonic kidney cells (HEK293T). In addition, receptor expression levels were monitored by western blotting. At the same time, basal as well as ligand-induced signaling was assessed by luciferase reporter gene assays.The results are as follows:1. Two pairs of primers of pig GHSR was designed based on the sequences deposited in GenBank. And the coding region of GHSR was amplified for the cloning with1101bp, encoding366amino acids. The cloned fragment was inserted into pcDNA3.1(+), a eukaryotic expression vector, by the T4DNA ligase to obtain the recombinant plasmid of pcDNA3.1(+)-myc/pGHSR. 2. Forward and reverse primers containing the mutation site were designed. The recombinant plasmid of pcDNA3.1(+)-myc/pGHSR was as the template for the site-directed mutagenesis of six mutations (P108L、I134T、V160I、C173R、A204D and F279L) of pGHSRs by Fast Mutagenesis System.3. HEK293T cells were transfected with the plasmids of WT and mutant pGHSRs by liposome transfection method, then extraction of total protein. And detection of total protein expression by western blotting, conducted by the monoclonal antibody of anti-myc and the IgG of goat anti-mouse. Results showed that the total expression of six mutations had reduced compared with pGHSR WT, the P108L and F279L showed a slight reduction, while the A204D had significantly decreased, and tne C173R showed the minimum expression.4. The plasmids of WT and mutant pGHSRs was transient transfected into HEK293T cells by lipofectamineTM2000respectively with the luciferase reporter gene vector pGL3to test the Ca2+level of cells, at the same time, we selected one kind of agonist and inverse agonist of GHSR to examine the change of the Ca2+level after stimulating by ligand. Results show that the pGHSR WT has a high level of constitutive activity, compared with WT, the constitutive activity of P108L, V1601and F279L variants had significantly decreased, while the C173R and A204E variants lacks constitutive activity. Stimulation of the WT receptor with Hexarelin, the Ca2+level increased with the increased concentration of Hexarelin. The I134T mutant, despite its high level of constitutive activity, fails to respond to Hexarelin stimulation. The C173R mutant also fails to respond to Hexarelin stimulation. On the contrary, the A204E variant that lacks constitutive activity, each of these mutants retained some degree of ligand-independent signaling. Stimulation of the WT receptor with SP-analog the inverse agonist, the Ca2+level reduced with the increased concentration of SP-analog. The P108L, I134T, V160I and F279L mutant were the same sa the WT, while the C173R and A204D fail to respond to SP-analog stimulation.