PMC

(A) Structures of Ca²⁺-free CaM (apoCaM) and Ca²⁺-bound CaM where the arrows indicate the Ca²⁺ binding sites. Image was reproduced from ref []. (B–C) CaM binding increases at the nucleus and Z-lines with higher [Ca²⁺]_i. Fluorescent CaM (60nM) was washed into permeabilized myocytes at 100 and 500nM [Ca²⁺]_i. Confocal images were taken to monitor CaM binding. After 40 min of fluorescent CaM wash-in (shown in B) fluorescence intensities were quantified at M-lines, Z-lines and nucleus (normalized to fluorescence intensity at M-lines at 100nM [Ca²⁺]_i) for [Ca²⁺]_i of 100 and 500 nM. Image reproduced from ref [] with permission. (D) Ca²⁺ regulates both functional populations of CaM: the dedicated CaM pool in complex with its target channels as well as the promiscuous CaM pool regulating CaMKII and CaN.

A. Control of tissue expression. Activity of β-gal of the targeting cassette under the regulation of endogenous regulation was detected by ONPG analysis on homogenates of the detailed tissues. The results from 3 CaM KMT^-/- and 2 CaM KMT^+/- 3 months old heterozygous male SVJ129 mice are presented. C57Bl6/J mice presented the same pattern. CaM KMT^+/- (HET) and CaM KMT^-/- (KO) B. Accumulation of hypomethylated CaM. Phosphoimage of lystaes of tissues and organs excised from CaM KMT^-/- (KO) or CaM KMT^+/+ (WT) adult females were incubated with HsCaMKMT and H³ Adomet. The control was recombinant CaM incubated with CaM KMT (C). The figure represents one of three experiments with different female mice of both SVJ129 and C57Bl6/J mice. The numbers at the left side of the figures represent the protein size marker.

(A) Newly-eclosed w¹¹¹⁸ flies stained for CaM (anti-Dictyostelium CaM: green), MyoV (red) and F-actin (blue). CaM is detected in rhabdomeres and cytoplasm, but prominently with MyoV. In the dark, CaM localizes prominently along the back of RTW cytoplasm and after 2 min light, CaM localizes to the rhabdomere base. (B) Dark-adapted and 2 min illuminated cam³³⁹/cam³⁵² heteroallelic ommatidia are stained for CaM (anti-Dictyostelium CaM: green), MyoV (red) and F-actin (blue). In cam³³⁹/cam³⁵² heteroallelic hypomorphs, CaM staining is weak and MyoV delocalised. In both dark and illuminated conditions, MyoV localizes at rhabdomere tips and is diffuse in the cytoplasm. A second anti-CaM antibody (from Dr. Beckingham) showed a similar staining pattern, but another anti-CaM antibody (from Dr. Klee) visualized mainly rhabdomeric CaM (). (C) An electron micrograph of a 2min illuminated cam³³⁹/cam³⁵² heteroallelic hypomorph ommatidium shows ommochrome pigment granules positioned behind organelle-poor RTW cytoplasm. Scale bar: 2 μm. (D) Co-immunoprecipitation of CaM and Mlc-c by myc-MyoV-CT. Lanes 1 and 2, extracts from w¹¹¹⁸ and Rh1gal4 >myc-tag MyoVCT heads, respectively. Lanes 3 and 4, anti-myc immunoprecipitants from w¹¹¹⁸ and Rh1gal4 >myc-tag MyoVCT heads, respectively. Neither CaM nor Mlc-c is co-immunoprecipitated from control w¹¹¹⁸ heads (lane 3). myc-tag MyoVCT immunoprecipitates both CaM and Mlc-c from Rh1Gal4>myc-MyoVCT heads (lane 4).

(A) GRK5 activity as a percent of basal activity (no CaM) for inhibitory effects and a percent of maximum activity (plus CaM) for activating effects (mean ± SD, n≥3). Data are presented as mean ± SD, n≥3.
(B) Surface representation of the full-length CaM/GRK5 model shown in . CaM binding (green) overlaps with the membrane-binding (blue dash) and receptor-binding (red dash) regions of GRK5.
(C) A cartoon of CaM and EF-hand mutants CaM 12 and CaM 34 with calcium shown as red dots.
(D) Peptide phosphorylation (top left), PC membrane binding (top right) and β₂AR phosphorylation (bottom) by GRK5 with increasing concentrations of CaM, CaM 12 and CaM 34. GRK5 activity is expressed as the fold change of basal activity (no CaM). Data are presented as mean ± SEM, n=3.
See also .