The overall goal of this procedure is to investigate the degradation pathway of the KISS one receptor, as well as the effect on receptor function of genetic mutations associated with human diseases. The first step is to introduce desired mutations in a MT tagged kiss one receptor by site directed mutagenesis, followed by transformation into e coli and sequencing to confirm introduction of the desired mutations. The next step is to transfect the Micks one receptor into human embryonic kidney cells to study degradation of the KISS one receptor using inhibitors of the lysosome and the proteasome.
Subsequently, the micki one receptor is immuno precipitated from treated cells using an aros conjugated anti mic ttag antibody. The final step of the procedure is the quantification of immuno precipitated KISS one receptor by western blot analysis. Ultimately, results can be obtained that revealed the degradation pathway of the receptor.
Alternatively, the effect of genetic mutations on receptor behavior can be determined as well. The techniques we're showing today can help scientists answer key questions in the G-protein couple receptor feud, such as how receptor function is regulated, as well as how genetic mutations in these receptors can eventually lead to the associated disease phenotypes in humans. The main Advantage of this lysis and western blatt technique over traditional methods is that this technique has been optimized to preserve the integrity of membrane proteins and maximize detection power.
The implications Of this technique extend toward therapy of human diseases associated with genetic mutations in the protein coupled receptors because the mechanism underlying the disease phenotype can be identified and characterized. The template for site directed mutagenesis of the S one R gene is the full CDNA sequence of the human KISS one R with a MT tag fused to its N terminus, which is cloned into a PCs two plus expression vector. Primers are designed to carry the desired mutation.
According to these guidelines, both forward and reverse. Primers must contain the desired mutation and anil to the same sequence on opposite strands of the plasmid. Primers should be 25 to 45 bases long and end in one or more, C or G bases introduced.
Mutations should be in the middle of the primer and flanked by about 10 to 15 bases of correct sequence on both sides. The melting temperature or TM of the primers should be greater than or equal to 78 degrees Celsius. The best amplification conditions are determined by adding A-P-C-R-X enhancer solution with or without DMSO, at least two concentrations of DMSO, such as 4%and 8%should be used in this determination.
This table shows the successful combination of reagents to mutate and amplify the GC Rich KISS one R.The cycling conditions of successful mutagenesis and amplification of GC Rich KISS one R are as follows, A two minute hot start followed by 18 cycles of 30 seconds melting at 95 degrees Celsius. One minute, an kneeling at 55 degrees Celsius and six minute extension at 68 degrees Celsius. Finally, an additional 10 minute extension at 68 degrees Celsius is added at the end of the last cycle at the completion of the PCR reactions.
Methylated parental DNA is eliminated by incubation of the amplification product with DPN one. The successful generation of the mutants is then checked by sequencing. In this study, degradation of the S one R is investigated in human embryonic kidney cells or HC 2 93, seated in four six well plates and transiently transfected with one microgram of total DNA per well samples are in triplicates to begin this procedure.
24 hours after transfection of HC 2 93 cells with Mki one R replaced the cell medium with one milliliter of DMEM containing 2.5%FBS. This decrease in serum may facilitate and or amplify the detection of results 16 hours before beginning the lysis procedure, add lysosome inhibitor directly to the 16 hour sample. Wells incubate at 37 degrees Celsius 10 hours later, which is six hours before lysis.
Add leptin to the samples for the six hour time point and continue incubating samples at 37 degrees Celsius to the samples. In two six well plates add freshly prepared proteasome inhibitor at the appropriate times. Incubate at 37 degrees Celsius.
Add vehicle to all wells of the fourth six. Well plate and incubate a 37 degrees Celsius for 16 hours. At the end of the incubation time, move all the plates to ice to perform cell lysis.
To prevent protein degradation, the entire lysis procedure must be performed on ice. First, aspirate the medium and wash cells once with one milliliter of ice cold phosphate buffered saline or PBS. Then at 100 microliters of ice cold lysis buffer containing protease inhibitors.
To each well remove cells with a cell scraper and transfer the cell lysates to centrifuge tubes. To increase protein yield, combine triplicates into one tube. Since the KISS one receptor is a membrane protein, it is important not to sonicate the cell lysates because sonication leads to aggregation of membrane proteins, which will not migrate properly.
During electrophoresis, Pass the cells about 10 times through a 20 gauge needle. Do not sonicate the samples. Next, incubate the cell lysates for one hour at four degrees Celsius on a rocking platform.
After one hour, centrifuge the cell lysates in a refrigerated centrifuge at four degrees Celsius for 10 minutes. At 10, 000 RPM, transfer the supernatants to new tubes without disturbing the pellets. After determining protein concentration dilute the cell lysates to one milligram per milliliter.
With lysis buffer containing protease inhibitors, the lysates can now be used for immunoprecipitation of mki one R, which will be shown next prior to immuno precipitating mki one R wash the appropriate amount of aros conjugated anti antibody twice with ice cold. PBS add 2.5 micrograms of agros conjugated anti antibody to 400 micrograms of lysate protein. Immuno precipitate the mikki one R on lysates overnight at four degrees Celsius on a rocking platform On the following morning, spin down agros speeds by pulse centrifugation at four degrees Celsius.
Aspirate and discard the supernatants without disturbing the pellets. Wash the beads once with ice cold lysis buffer. After that, wash twice with ice cold.
PBS aspirate the PBS and resuspend the beads containing antibody bound mic KISS one R in sample loading buffer to prepare samples for western blot detection of Mick KISS one R immuno complexes heat sample buffer containing beads for 30 minutes at 37 degrees Celsius. After 30 minutes, move the tubes immediately onto ice for five minutes. Separate proteins by electrophoresis in a four to 15%gradient gel.
Next, transfer the proteins at 25 volts for 30 minutes in transfer buffer to an IM and FL PVDF membrane for infrared detection. When the transfer is complete, wash the membrane for five minutes at room temperature with tris buffered saline or TBS then block with Lycor odyssey blocking buffer for one hour at room temperature on a rocking platform. Next, incubate the membrane overnight at four degrees Celsius with rabbit EC antibody in blocking solution containing 0.1%tween 20 on the following day, remove the primary antibody and wash the membrane three times for five minutes each with TBS containing 0.1%between 20.
After the final TBST wash at goat infrared dye 800 CW labeled anti REIT IgG in blocking buffer containing tween. 20 and SDS incubate for one hour at room temperature. At the end of the one hour, remove the secondary antibody and wash the membrane three times for five minutes each with TBST and the last time with TBS only to remove the remaining twin 20.
The Micks one R on the PVDF membrane will be imaged using the Lycor Odyssey infrared imager. To begin place the membrane on the bottom left corner of the scanner, aligning it with the grid cover with the rubber mat, smooth out bubbles with a roller and close the lid. Create a new project file on the computer.
Name the file, click done, and then enter the scanner. Login on scan size, the scanner console box to fit your membrane and then choose 169 micrometer resolution and medium image quality. Choose intensity settings for the 700 and 800 channels according to the expected strength of each signal.
This is for signal visualization purposes only and will not influence quantification. After naming and saving the scan, click okay to open it in a new window For quantification MKI one R monomers should be visible at approximately 43 kilodaltons using the box tool On the left hand sidebar, draw a box around the first band, drag the box around to make sure all bands fit in. Then copy and to paste the box over all the bands.
Select all boxes using control A then select the option to subtract median background. Click report on the top menu and a spreadsheet with the quantification values will appear. Numbers here represent the quantification of gel bands in arbitrary units.
The results of optimizing PCR conditions to facilitate the amplification of a range of KISS one R mutants are shown in this image of a 1%AROS gel stained with AUM bromide and visualized using UV light. Plasmid Bands of six kilobases are visible in both lanes. Loaded with PCR products amplified in the presence of 4%and 8%DMSO, but not in the first lane, which was loaded with a PCR product amplified in the absence of DMSO.
The Mick KISS one R construct was subsequently used for analysis of KISS one R degradation and the results are shown here. HEC 2 93 cells expressing Micks one R were treated with leptin for zero, six, or 16 hours or MG 1 32 for 0 2 4 6 or 16 hours, followed by cell lysis, immunoprecipitation and western blot analysis. The top panel shows the scan of MKI one R monomers, and the bottom panel shows the quantification of those bands.
Results are represented as fold increase over untreated cells. Quantification of bands indicates that six hours or 16 hours of leptin treatment did not affect M KIS one R protein levels. Conversely, treatment with MG 1 32 resulted in a time dependent increase in M KIS one R protein, which culminates in a 45 fold accumulation of the receptor after 16 hours of incubation with MG 1 32.
These observations indicate that unlike most G protein coupled receptors, KIS one R is degraded by the proteasome rather than the lysosome. After watching this video, you should have a good understanding of how to successfully perform site directed mutagenesis of the C reach templates. You should also have a good grasp on how to detect lysosomal and proteasomal degradation of membrane proteins by immunoprecipitation and Western blood analysis After the development.
These techniques paved the way for investigators working with G-Protein couple receptors to explore the regulation of receptor signaling in functional assays in vitro. They also allowed researchers to examine the resultant effect on receptor function of genetic mutations in these receptors that are associated with human diseases.
