Calcium Signaling: Emanuel E. Strehler

The Na+/H+-exchanger regulatory factor-2 (NHERF2) anchors the plasma membrane calcium pump isoform PMCA2w/b to the apical membrane in polarized MDCK cells. Confocal fluorescence images are shown of the apical membrane of MDCK cells co-expressing GFP-PMCA2w/b (green, A1) and NHERF2 (red, A2), and stained for actin (blue, A3). Panel A4 is a merged image, where white indicates overlap of the green, red and blue staining.

Illustration B shows anchorage of PMCA2w/b to the apical actin cytoskeleton by NHERF2 via ezrin. PMCA2w/b binds via its C-terminal ETSL sequence to the PDZ protein NHERF2, which interacts through its ERM domain with ezrin.

Source: Padanyi R, et al. Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells. The Journal of Biological Chemistry. 2010;285:31704.

Stable expression and function of PMCA2w/b in MDCK cells. Image A (top) shows MDCK cells stably expressing PMCA2w/b co-stained with antibodies against PMCA2 (green), Na+/K+-ATPase (blue) and ezrin (red). A substantial overlap of the fluorescence signals for PMCA2 and ezrin is observed on the apical side (merged image on bottom). Cells were analyzed by confocal laser scanning microscopy with vertical z-scans.

Image B (bottom) shows the time course of Ca2+ transients measured in empty vector control or PMCA2w/b expressing MDCK cells transfected with the genetically encoded Ca2+ indicator GCaMP2. The arrow indicates administration of 100 micrometer ATP to elicit a Ca2+ spike. Twenty to 30 cells were measured in a single experiment and the experimental data were fitted using GraphPad Prism4 software. This experiment demonstrates that expression of PMCA2w/b dramatically shortens the duration of the Ca2+ signal compared to control cells that express mainly PMCA4b.

Source: Antalffy G, et al. Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells. Biochemical and Biophysical Research Communications. 2011;410:322.

Myo10 consists of two heavy chains with an N-terminal motor domain; a "neck" region of 3 IQ domains involved in light chain binding; a putative coiled-coil region; and a tail region containing pleckstrin homology (PH) domains, a myosin tail homology (MyTH) domain and a C-terminal FERM (4.1, ezrin, radixin, moesin) domain.

The IQ motifs bind calmodulin (CaM) or calmodulin-like light chains, which regulate Myo10 activity and Ca2+ sensitivity. Upon expression of CALML3 (in the presence of Ca2+), CALML3 competes successfully with CaM and binds tightly to IQ3 and possibly IQ1 and IQ2 of Myo10. CALML3 binding results in increased Myo10 expression and function, as demonstrated by increased filopodial extension and enhanced directional cell migration.

Sources: Caride AJ, et al. Kinetic analysis reveals differences in the binding mechanism of calmodulin and calmodulin-like protein to the IQ motifs of myosin-10. Biochemistry. 2010;49:8105, and Strehler EE. Emanuel Strehler's work on calcium pumps and calcium signaling. World Journal of Biological Chemistry. 2011;2:67.

Sections from normal skin (top) and basal cell carcinoma (bottom) stained for CALML3 (left) and Ki-67 (right). Note the polarized expression of CALML3 throughout the normal epidermis with strong nuclear staining in the upper cell layers (black arrows in top left panel). Ki-67 labeling is sparse and confined to the basal cell layer (black arrows in top right panel).

In contrast, the highly proliferating basal cell carcinoma (bottom panels) shows strong CALML3 downregulation in the tumor (white arrowhead), but normal CALML3 expression in the overlying epidermis.

CALML3 staining is increased in basal and suprabasal cells immediately adjacent to the wound bed (white arrows) and concentrated in the cell periphery where it may activate Myo10. CALML3 is thought to be involved in normal epithelial cell differentiation, and its absence is a hallmark of many cancers.

Source: Bennett RD, et al. Calmodulin-like protein upregulates myosin-10 in human keratinocytes and is regulated during epidermal wound healing in vivo. The Journal of Investigative Dermatology. 2009;129:765.

Micrograph of a HeLa cell expressing green fluorescent protein (GFP) tagged myosin-10 together with calmodulin-like protein. Note the abundant fluorescent myosin-10 puncta in filopodial extensions. Image taken by Richard D. Bennett (Mayo Graduate School PhD student, Molecular Biology track)

The pump is shown in the autoinhibited state, in which the calmodulin-binding domain (CaM-BD) binds to intramolecular sites on the first and second hydrophilic loops. The ten membrane-spanning domains, the catalytic phosphorylation site (~P) and the ATP-binding site (AS) are indicated. The PDZ domain-binding region present in some calcium pump splice variants is marked by a circled PDZ. Arrows point to the regions (“hotspots” A and C) where isoform diversity is created by alternative RNA splicing. For details, see Strehler, E.E. and Treiman, M., Curr. Mol. Med. 4:323-335 (2004).

Alternative splicing affects the membrane localization of the plasma membrane calcium pump (PMCA) isoform 2b. Full-length PMCA2w/b, PMCA2x/b, and PMCA2z/b (constructs schematically shown on top) were expressed in polarized MDCK (dog kidney epithelial) cells, and the localization of the pumps was studied by confocal fluorescence microscopy. PMCA2 was detected with an isoform-specific antibody followed by an Alexa-488 labeled secondary antibody (green). Nuclei were stained with DAPI. PMCA2w/b shows apical localization in addition to basal and lateral staining (left panel), whereas PMCA2x/b and PMCA2z/b are confined to the basal and lateral membranes (middle and right panel). Corresponding x:z sections are shown below the en face views. Arrows indicate the height at which the en face sections were captured. Bar, 20 µm. For details, see Chicka, M.C. and Strehler, E.E., J. Biol. Chem. 278:18464-18470 (2003).