em A /em ) Representative histogram overlays (see key) of Alexa-fluor 647 signals from total PI-negative populations of anti-HA immunostained mock-transfected HEK293T/17 cells (black dotted line), no-antibody (non-stained) control (grey-shaded) and immunostained (red line) WT 11.2 samples and immunostained 11.2 mutants Q1655A (blue line), I1654A (brown line), Y1649A (gold line) and K1647A (green line). in current density. The larger decrease in current density as compared to surface expression imparted by these 11.2 subunit mutations hints at the possibility that -actinin not Rabbit Polyclonal to RHO only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity. 0.05 was considered statistically significant (*). RESULTS Yeast Two-hybrid Screening We previously showed co-immunoprecipitation of -actinin with 11.2 and determined that a C-terminal region in 11.2 spanning AAs 1588-1670 is required for its interaction with -actinin (32). This 11.2 region also encompasses the IQ motif (AAs 1644-1670) where calmodulin binds to facilitate Ca2+-dependent inactivation (41, 42). To better define the region in -actinin required for this interaction and to test whether this interaction is direct, we used the yeast two-hybrid (Y2H) system (62). We designed a series of Y2H expression constructs covering the different polypeptide regions in -actinin 1 (Figure 1A). Out of eleven -actinin constructs, only the SR4EF and SR234EF polypeptides consisting of the C-terminal four EF hands preceded by either one (SR4EF) or three (SR234EF) spectrin repeats of -actinin-1 Laninamivir (CS-8958) showed interactions with the 11.2 AAs 1506-1871) (Figure 1B). SR234EF gave consistently strong interaction readouts and was thus used for all subsequent interaction assays. Open in a separate window Figure 1 Defining -actinin-1 – 11.2 interacting regions by yeast two-hybrid assayTop: Schematic of 11.2 bait encoded by a cDNA for the C-terminal region of 11.2 spanning AAs 1506-1871 (red frame), which was subcloned in frame with the Gal4 DNA-binding domain in the Y2H bait expression vector pGBKT7 for initial identification of the -actinin-1 binding region. Laninamivir (CS-8958) Bottom: Schematic of -actinin-1. The optimal prey polypeptide encompassing spectrin repeats 2C4 and EF hands (SR234EF; AAs 391-892) of -actinin-1 is depicted by the red frame. and the -actinin-1 SR234EF core (AAs 391-892) was subcloned and expressed as an MBP-tagged fusion protein. Purified GST-tagged WT and mutant 11.2 IQ motifs were immobilized on glutathione-Sepharose and equal amounts of purified MBP–actinin-1 were added. In contrast to the ability of immobilized WT 11.2 IQ-GST to pull-down recombinant -actinin-1, equal amounts of the K1647A, Y1649A and I1654A-GST fusion proteins exhibited only minimal amounts of pull-down of the -actinin target and GST alone no detectable pull-down of (Fig. 3). Open in a separate window Figure 3 Pull-down assay using glutathione-Sepharose-bound WT and point-mutant 11.2 C-terminal polypeptides as bait to capture purified MBP–actinin-1The indicated WT and point-mutant 11.2 C-terminal polypeptides spanning AA1576-1733 were expressed as GST-fusion proteins and immobilized on glutathione-conjugated Sepharose (illustrates that comparable amounts for each the GST – 11.2 fusion polypeptides were present. Similar results were obtained in two other experiments. Under basal conditions when Ca2+ is low apoCaM pre-associates with the IQ motif (63, 64) and could mediate Laninamivir (CS-8958) potential effects of point mutations within the IQ motif. To evaluate whether these mutations affected binding of apoCaM we utilized a fluorescence polarization (FP) assay we had adopted earlier for defining CaM binding (52) to evaluate the relative binding-affinities of the WT and mutant 11.2 IQ domains for apoCaM (Fig. 4; Table 1). Accordingly, only the I1654A but not K1647A or Y1649A mutation affected apoCaM binding, in perfect agreement with earlier work (64). The F1648A and Q1655A also did not affect apoCaM binding, again in full agreement with previous work (64). Open in a separate window Figure 4 Titration of a11.2 IQ domain.Indeed, analysis of the behavioral and biochemical effects of nifedipine and BayK8644 in knock-in mice harboring the 11.2 Thr1066Tyr mutation that makes CaV1.2 insensitive to these drugs strongly indicates that abnormal CaV1.2 activity can cause neurotoxicity and is a major underlying factor in anxiety disorders, depression, and self-injurious behavior (7). of CaV1.2 that individually impaired -actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 channels composed of the corresponding -actinin-binding-deficient mutants results in a 35C40% reduction in surface expression compared to wild-type channels. Moreover, the mutant CaV1.2 channels expressed in HEK293 cells exhibit a 60C75% reduction in current density. The larger decrease in current density as compared to surface expression imparted by these 11.2 subunit mutations hints at the possibility that -actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity. 0.05 was considered statistically significant (*). RESULTS Yeast Two-hybrid Screening We previously showed co-immunoprecipitation of -actinin with 11.2 and determined that a C-terminal region in 11.2 spanning AAs 1588-1670 is required for its interaction with -actinin (32). This 11.2 region also encompasses the IQ motif (AAs 1644-1670) where calmodulin binds to facilitate Ca2+-dependent inactivation (41, 42). To better define the region in -actinin required for this interaction and to test whether this interaction is direct, we used the yeast two-hybrid (Y2H) system (62). We designed a series of Y2H expression constructs covering the different polypeptide regions in -actinin 1 (Figure 1A). Out of eleven -actinin constructs, only the SR4EF and SR234EF polypeptides consisting of the C-terminal four EF hands preceded by either one (SR4EF) or three (SR234EF) spectrin repeats of -actinin-1 showed interactions with the 11.2 AAs 1506-1871) (Figure 1B). SR234EF gave consistently strong interaction readouts and was thus used for all subsequent interaction assays. Open in a separate window Figure 1 Defining -actinin-1 – 11.2 interacting regions by yeast two-hybrid assayTop: Schematic of 11.2 bait encoded by a cDNA for the C-terminal region of 11.2 spanning AAs 1506-1871 (red frame), which was subcloned in frame with the Gal4 DNA-binding domain in the Y2H bait expression vector pGBKT7 for initial identification of the -actinin-1 binding region. Bottom: Schematic of -actinin-1. The optimal prey polypeptide encompassing spectrin repeats 2C4 and EF hands (SR234EF; AAs 391-892) of -actinin-1 is depicted by the red frame. and the -actinin-1 SR234EF core (AAs 391-892) was subcloned and expressed as an MBP-tagged fusion protein. Purified GST-tagged WT and mutant 11.2 IQ motifs were immobilized on glutathione-Sepharose and equal amounts of purified MBP–actinin-1 were added. In contrast to the ability of immobilized WT 11.2 IQ-GST to pull-down recombinant -actinin-1, equal amounts of the K1647A, Y1649A and I1654A-GST fusion proteins exhibited only minimal amounts of pull-down of the -actinin target and GST alone no detectable pull-down of (Fig. 3). Open in a separate window Figure 3 Pull-down assay using glutathione-Sepharose-bound WT and point-mutant 11.2 C-terminal polypeptides as bait to capture purified MBP–actinin-1The indicated WT and point-mutant 11.2 C-terminal polypeptides spanning AA1576-1733 were expressed as GST-fusion proteins and immobilized on glutathione-conjugated Sepharose (illustrates that comparable amounts for each the GST – 11.2 fusion polypeptides were present. Similar results were obtained in two other experiments. Under basal conditions when Ca2+ is low apoCaM pre-associates with the IQ motif (63, 64) and could mediate potential effects of point mutations within the IQ motif. To evaluate whether these mutations affected binding of apoCaM we utilized a fluorescence polarization (FP) assay we’d adopted previously for determining CaM binding (52) to judge the comparative binding-affinities from the WT and mutant 11.2 IQ domains for apoCaM (Fig. 4; Desk 1). Accordingly, just the I1654A however, not K1647A or Y1649A mutation affected apoCaM binding, in ideal agreement with previously function (64). The F1648A and Q1655A also didn’t have an effect on apoCaM binding, once again in full contract with previous function (64). Open up in another window Amount 4 Titration of a11.2 IQ.
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