Previous research has shown that β1A integrins form a heterogeneous group of two predominant bands of about 120kDa and 140kDa size when analysed by immunoblotting (Bottcher et al., 2012;
Meng et al., 2005; Tiwari et al., 2011). The different apparent weights are caused by distinct glycosylation patterns in these two groups. Thus, the higher weight population exhibits either more, or altered glycosylation pattern of N-glycosylated integrins. However, various interpretations about the influence of integrin location and activity have been made in an attempt to explain the two molecular weights. The upper 140kDa band is often referenced as
“mature”, in contrast to the lower, 120kDa immature or precursor band. Despite their name, it is not clear whether each group is composed of one, or several β1A integrin populations diverging in subcellular locations, glycosylation levels, affinities for α-integrin association and activity states. Interestingly, not only the upper, mature band but also the faster migrating lower band can contain integrin populations from the cell membrane (Meng et al., 2005). Additionally, the 140kDa population includes inactive β1A integrins from Golgi and the lower integrin band is posted to include inactive β1A integrins from the ER (Tiwari et al., 2011). Little is known about surface β1A integrins weight pattern contribution of surface integrins present outside adhesions compared to integrins incorporated in adhesion, not to mention the pattern differences between FA and FB integrins. The second scope of weight pattern analysis is possible by β1A band abundancy comparisons, which help interpret integrin-recycling and kindlin binding properties (Bottcher et al., 2012; Margadant et al., 2013). Previous studies demonstrated in pulse-chaise experiments, that the upper, integrin band is created via integrin maturation processes from synthesized 120kDa precursor integrins but also through integrin recycling of 140kDa integrins, which have passed the internalization and membrane reintegration process, as high glycosylated 140kDa integrins. In other words, the upper band’s intensity can be increased either by increasing overall β1A integrin synthesis or by blocking β1A terminal degradation. These two alternatives can be experimentally distinguished by analysing the total β1A expression (sum of 120kDa and 140 kDa band) and relate them to the ratios of 140/120kDa integrin quantities. Total protein expression numbers might increase in both cases, once by having cells produced more integrin in total and once by degrading less integrin (Figure 4.5C). However, only if integrin degradation is inhibited the mature/precursor integrin ratio (140/120 kDa) would increase by keeping the mature integrins in the membrane retargeting cycle (Figure 4.5D), in this case the 140kDa numbers would increase due to freshly synthesized 120kDa precursor integrins, and their maturation and membrane integration. Furthermore, (Margadant et al., 2013) outlined by WB and FACS analysis
47 how defective kindlin-binding to β1A integrins reduces the expression of mature, high glycosylated β1 integrins on the surface. Having one FB β1A mimicking mutant (K/Q) and one FA
mimicking mutant (K/R) at our disposal, we investigated if β1A integrins show different glycosylation and recycling patterns due to their distinct acetylation states (Figure 4.5). Equal amounts of GD25 nt or GFP tagged β1A wt / K/R and K/Q expressing cells were plated in petri dishes and grown to confluence in a 4.5G cell medium. For drug experiments, SBHA was administered to the cell medium o/n before cell lysis. WB analysis for integrin heterogeneity was performed by blotting for GFP. It revealed two bands of β1A wt integrins in GD25 cells at 130kDa and 170kDa. The shift to the above defined 120kDa and 140kDa bands can be explained by the linked GFP protein, which increases the integrin weights. Strikingly, the acetylation mimetic and FB like K/Q integrin mutant was expressed to a higher extend (Figure 4.5A,C) in the cell and had an increased mature/precursor ratio (Figure 4.5A,D) compared to β1A wt. This complies with the theory that constitutive presence of an acetylation signal, which is here mimicked by the K/Q mutation, blocks the concurrent ubiquitination and subsequent protein degradation. That way β1A K/Q integrins increase in total and continuously stay in a membrane integration/internalization/reintegration cycle. In contrast, GD25 cells expressing β1A K/R
Figure 4.5- Acetylated β1A integrins are mature and highly glycosylated: An equal number of nt or stably expressing and GFP tagged β1A GD25 cells were plated in dishes and grown until confluence in 4.5G media.
Subsequently, cells were lysed and the same volumes loaded on 8% SDS gels under non-reducing conditions.
For SBHA treatments, SBHA was added to the cell medium the night before lysis. Membranes were stained for GFP (rabbit pAb). A) Representative GFP stainings of β1A wt, K/R and K/Q integrins, mature (170kDa) and precursor (130kDa) bands a visualized. Intensities of mature (170kDa) and precursor (130kDa) bands were quantified by ImageJ originating from three blots for acetylation mimetic mutants and two repeats for SBHA treatments. C) Mean values and SEMs plus single values of band intensity sums are blotted as relative values to β1A wt. D) shows the ratios of mature/precursor (170/130kDa) intensities relative to β1A wt including single measurements, their means and SEMs.
A B
C D
48 integrins did not have reduced total integrin numbers, but presented less mature integrins than the β1A wt control cells. Since a lysine to arginine mutation mimics the deacetylated lysine state but also was demonstrated to prevent ubiquitination and protein degradation (Lu et al., 2013;
Trovato et al., 1999), we conclude that ostensibly a deacetylated K794 residue is sufficient for β1A
integrin to avoid recycling and embark on the degradation pathway after internalization. Due to the inability to bear ubiquitination by the arginine mutant itself, integrin degradation might be put into execution by ubiquitination of neighbouring K774/K784 and K798, which may be triggered through the β1A deacetylation signal, mimicked with arginine.
As K/Q integrins are mimicking the acetylation state, we wondered whether we could reproduce an equal heterogeneity pattern by increasing β1A wt acetylation via the KDAC inhibitor SBHA (Figure 4.5B). This drug approach managed to mimic the increased β1A expression and the positive mature/precursor ratio like with β1A K/Q integrins. We remind, that in contrast to our former mutant-based strategy, a drug approach does not obstruct the integrin’s choice of PTM signal on lysine K794, which allows the lysine therefore to be acetylated, deacetylated but also ubiquitinated. With the knowledge about the specific targeting capacities of SBHA (Figure 4.18) on β1A acetylation levels, our findings suggest that SBHA mediated β1A integrin acetylation may block degradation, also by winning the battle against ubiquitination, the degradation signal. These results might suggest that β1A K794 acetylation (K/Q mutant) increases β1A glycosylation and supports integrin recycling to the plasma membrane but blocks β1A integrin degradation (Figure 4.5). Deacetylated β1A integrins (K/R, K/A mutants) have problems to mature und to get expressed on the cell membrane (Figure 4.5). K/R integrins might bind less kindlin whereas K/Q might be capable of binding more kindlin, which is that way supporting β1A K/Q integrin maturation and cell membrane integration (compare total surface integrin expression Figure 4.9B). In this
integrin surface expression fraction
1A wt
1A K/R
1A K/Q
1A K/A
0.0 0.5 1.0
1.5 sns
sns sns
Figure 4.6 – Compromised plasma membrane insertion for K/A and K/R β1A integrins: FACS analysis of surface and total β1A integrin. Stable GD25 β1A wt, K/R, K/Q and K/A GFP cells were stained in 4.5G for total membrane bound β1A integrin with TS 2/16 antibody. Total integrin expression was measured by GFP values.
Surface (TS 2/16) to total (GFP) integrin ratios are depicted as mean and SEM of four to seven experiments.
Applied statistical test: Kruskal-Wallis Test with Box-and Whisker blot notches, CI 95% in Statgraphics.
49 experiment whole cell lysates were analysed. Including ER and Golgi integrin populations, the discussed WB analysis has no informative value about net surface integrin expression. That is why a complementary surface integrin study by FACS analysis was performed that certified a restrained plasma membrane insertion for deacetylated integrins but no statistical significant changes of plasma membrane insertion capacities of K/Q compared to β1A wt integrins at 4.5G levels (Figure 4.6). This result shows that the increase in mature integrins is not raising the total membrane expression levels compared to β1A integrin.