2.2 Methods
3.1.1 Qualitative assessment of β 1 -adrenoceptor phosphorylation . 59
β
1-adrenoceptor phosphorylation
Phosphorylation can be assessed both qualitatively as well as quantitatively. Quali-tative methods include radioactive phosphorylation assays, phosphosite-specific monoclonal antibodies in western blotting and qualitative mass spectrometry.
Radioactive phosphorylation
Radioactive phosphorylation assays visualize the amount of protein phosphoryla-tion using radioactive32phosphorous. To first set the experimental conditions, the concentration of fetal calf serum the stably transfected HEK293 cells were cultured in was tested. Figure3.1depicts this test. Four different FCS concentrations were used ranging from 0.25 % up to 10 % of FCS as medium supplement. The cells were stimulated with 100µM norepinephrine for five minutes. This induced receptor phosphorylation. Since the largest differences between the unstimulated and the stimulated conditions were observed in 1 % FCS, all subsequent experiments were performed using 1 % of FCS in the cell culture medium (Figures3.2and3.12).
The radioactive phosphorylation assay further shows that theβ1-adrenoceptor is phosphorylated in its basal state and that its phosphorylation increases upon adrenergic stimulation (Figure3.2).
Figure 3.1:Supplementing FCS in cell culture medium impacts ADRB1 phosphorylation.
(A) Radioactive phosphorylation assay with HEK293 cells stably overexpressing the β1-adrenoceptor. The cells were cultivated in FCS concentrations ranging from 0.25 % to 10 %. After incubation with32P an immunoprecipitation against theβ1-adrenoceptor was performed and the radioactive western blot was exposed on a film for three weeks.
Radioactivity was then measured using a phosphoimaging system. (B) Quantification of (A). n = 1
Figure 3.2:The β1-adrenoceptor is basally phosphorylated and its phosphorylation in-creases upon norepinephrine stimulation.
(A) Upper panel: Radioactive phosphorylation assay using unstimulated and stimulated HEK293 cells stably overexpressing theβ1-adrenoceptor. Lower panel: Western blot of the radioactive blot, to control for equal protein loading. (B) Quantification of (A). n=3
3.1.2 Purification of the β
1-adrenoceptor
In order to determine the phosphorylation pattern of theβ1-adrenoceptor via mass spectrometry, the receptor first needed to be purified. The purified receptor could then be separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the two receptor bands were cut out of the gel and underwent mass spectrometric analysis. The ADRB1 usually displays a two-band pattern on an SDS-PAGE and western blot (Figure3.3(A) - (C)), with one band at 55 kDa and the other at 68 kDa. It is proposed that this double band pattern is due to glycosylation and N-terminal cleavage (Hakalahti et al.,2013).
During a conventional immunoprecipitation the precipitating antibody is degraded together with its precipitated target. This leads to the degradation of the antibody into its light and heavy chain. The molecular weights of both moieties are 25 and 50 kDa, respectively. With the β1-adrenoceptor as antigen, this leads to a very prominent antibody band at nearly the same height as the lower band of the ADRB1 (Figure 3.3(A)). While this is not problematic in a western blot, as anti-light chain antibodies can be used for detection, this is not feasible in a gel that is designated to be further analyzed by mass spectrometry. To circumvent this problem and to ensure a higher purity of the immunoprecipitated ADRB1 a crosslink immunoprecipitation was performed. 1-15 mg of total protein was used in the crosslink immunoprecipitations in order to maximize the amount of ADRB1 protein available for MS analysis. The crosslink immunoprecipitation products were then separated on an SDS-PAGE gel and underwent western blotting (Figure3.3 (B)) and silver staining (Figure3.3(C)), respectively.
Silver staining was performed to visualize theβ1-adrenoceptor after purification.
After the staining the two ADRB1 bands were cut out off the gel, digested and analyzed via mass spectrometry. On a western blot the untransfected negative control was very clean as seen in Figure3.3(B). This shows the robustness of the assay, i.e. the antibody crosslinking seemed to be very effective, with no co-elution of the antibody with the desired antigen. In the silver staining, however, a very prominent band appeared at approximately 55 kDa. This band looked very similar to the lower band in the stably transfected HEK293 cells expressing the ADRB1.
This led to the conclusion that the upper band corresponded to the upper band of the ADRB1, but the lower band seemed to be a mixture of unknown proteins and the lower band of the ADRB1. Subsequent MS analysis suggested that this band consisted mostly of tubulin.
As the silver staining turned out to be relatively clean, apart from the band at 55 kDa there were no other unexplained bands, the in-gel digestion was
dis-continued in favor of an in-solution digestion of the immunoprecipitation eluate.
This means that the whole immunoprecipitation product was used for the mass spectrometry scans and not just the bands, that were cut out of the silver gels.
Figure 3.3:Immunoprecipitation and silver staining of theβ1-adrenoceptor.
(A) Purification of the ADRB1 using conventional immunoprecipitation. Untransfected HEK293 cells were immunoprecipitated as a negative control. Due to the antibody degradation into the heavy and light chain in SDS, the antibody band at 50 kDa is very prominent. (B) Crosslink immunoprecipitation. Untransfected HEK293 cells were immunoprecipitated as a negative control. The three lanes containing immunopre-cipitated ADRB1 represent different elution steps after the crosslink IP. (C) Purified β1-adrenoceptor from a crosslink immunoprecipitation was separated on an SDS gel and then silver-stained to assess the amount of purified receptor. An immunoprecipita-tion from untransfected HEK293 cells was used as negative control. To ensure that no antibody was co-eluting together with the ADRB1, additionally denatured IgG was used as a control. Ab = antibody
Qualitative mass spectrometry
In order to determine theβ1-adrenoceptor’s phosphorylation pattern, a qualitative mass spectrometry approach was implemented. To elucidate which amino acid residues on the ADRB1 can actually become phosphorylated, HEK293 cells sta-bly expressing theβ1-adrenoceptor were stimulated with 100µM norepinephrine for five minutes to induce maximal activation of the receptors and thus maximal phosphorylation of the receptor itself. Figure3.4depicts the workflow of sample preparation for MS analysis.
Figure 3.4:Workflow of sample preparation for mass spectrometry.
IMAC = immobilized metal ion affinity chromatography, IP = immunoprecipitation, NE = norepinephrine
The cells were lysed in a 3 % NP-40 lysis buffer and the lysates were crosslink-immunoprecipitated. The IP products were then reduced, alkylated and protease digested. Since the ADRB1 proved to be difficult to digest with trypsin alone, three different proteases were used: LysC combined with trypsin and chymotrypsin. The resulting mixture of peptides underwent an IMAC phosphopeptide enrichment to increase the abundance of the phosphorylated species over its unphosphorylated counterpart and was subsequently analyzed in the mass spectrometer.
The resulting phosphorylation pattern of theβ1-adrenoceptor after mass spectro-metric analysis is shown in Figure3.5.
Figure 3.5:Qualitative assessment of β1-adrenoceptor phosphorylation by mass spectro-metry.
Phosphorylation pattern of theβ1-adrenoceptor. Amino acids marked in white and black indicate phosphorylation under basal conditions and after stimulation with 100µM norepinephrine for 5 min, respectively.
Marked are all serines and threonines, which could be confidently identified as
Serine 312 and serine 412 have been previously described to be phosphorylated by protein kinase A and B, respectively. All other sites were newly identified. Data from five measurements were pooled to get an overview of the maximal phosphorylation pattern of theβ1-adrenoceptor.