Organization of h-CaBPs genes. The coding regions are shown in black boxes, and noncoding regions of exon 1 and exon 6 are shown in white boxes. Introns are shown as thick lines. A, organization of the h-CaBP1 gene. The gray outlined box indicates the position of the closest exon specific to the caldendrin gene. The numbers below CaBP1 indicate the position of the exon/intron junctions in the CaBP1 cDNA. B, organization of h-CaBP3 and h-CaBP5 genes. Gray boxes indicate the position of exons in nonoverlapping complementary strands. The gray outlined box indicates the extra exon (exon 3B) of the CaBP3 gene that is part of exon 3 in the CaBP5 gene. Arrows indicate selected primers (K, forward primers; k, reverse primers) used to amplify the introns. Numbers below CaBP3 and above CaBP5 indicate the position of the exon/intron junctions in the CaBP3 and CaBP5 cDNAs. C, agarose-gel separation of PCR products for CaBP3 and CaBP5 gene analysis. Selected PCR products (indicated by arrows) generated with primers indicated above the gel, were cloned or purified and partially sequenced to determine the organization of the CaBP3 and CaBP5 genes.

Immunolocalization of CaBP1 and CaBP5 in mouse retina. CaBP1 (UW72) (A) and CaBP5 (UW89) (B) expression is prominent in cell bodies located in the INL. CaBP1 expression is restricted to a small subset of INL neurons, while CaBP5 is expressed by a majority of INL neurons. No immunolabeling is visible in sections of mouse retina incubated in pre-immune sera from UW72 (C) or UW89 (D). Preincubation of UW72 sera with purified CaBP1 (600 nm) abolishes CaBP1 immunolabeling (E).Preincubation of UW89 sera with purified CaBP5 (600 nm) abolishes CaBP5 immunolabeling (F). Sections of mouse retina were double-labeled with antibodies to PKC and either CaBP1 (G) or CaBP5 (H). PKC immunolabels rod bipolar cell bodies and their processes (green). Very few CaBP1-labeled cells (red) are PKC-positive. In contrast, most CaBP5-positive cells (red) are double-labeled by antibodies to PKC (arrowheads, H), confirming their identity as rod bipolar cells. Cone bipolars are labeled by antibodies to CaBP5 but not PKC. Magnification bar: A–F, 50 μm; G–H, 25 μm.

Specificities of anti-CaBP antibodies and purification of retinal CaBPs. A, Ca²⁺-dependent mobility change in SDS-PAGE for partially purified CaBP1 (purified on DEAE-cellulose; see “Materials and Methods”), CaBP2-His₆ purified on Ni²⁺-chelex column, CaBP5-His₆ purified on Ni²⁺-chelex column. B, immunoreactivity of CaBP1, CaBP2, and CaBP5 with UW75 (raised against the common domain of CaBP), UW92 (raised against CaBP2), and UW89 (raised against CaBP5). C, retinal CaBPs were purified as described under “Materials and Methods” and blotted on polyvinylidene difluoride. The protein bands were digested with trypsin, and peptides were separated on a reverse phase column. The aa sequence of peptides was obtained using a combination of Edman degradation of matrix-assisted laser desorption/ionization (MALDI; see “Materials and Methods”). In brackets are the predicted residues from the cDNA sequence and the N-terminal side of tryptic digestion: x represent an undetermined aa, dotted points show that the sequence did not reach the end of the peptide. cont, contaminants from UW72. D, Western blot of retinal CaBPs probed with UW72 in the presence or absence of 100 μm [Ca²⁺].

Activation of CaM kinase II by CaBPs and interaction of CaBP2 with the peptide derived from the CaM-binding domain of CaM kinase II. A, the activity of purified CaM kinase II was measured in the presence of 2 μm Ca²⁺-binding proteins using syntide-2 as a substrate (Table I). The short forms CaBP1 and CaBP2 were used in the assays. B, binding of CaBP2 to the peptide derived from the CaM-binding domain of CaM kinase II. The peptide 290LKKFNARRKLKGAILTTMLA309 was coupled to CNBr-activated Sepharose, and CaM and CaBP2 were added in the presence of 50 mm Ca²⁺. The column was washed with 10 mm BTP, pH 7.5, containing 100 mm NaCl. Bound proteins were eluted with 1 mm EGTA. CaBP1 and CaBP5 did not bind to this column.

Model of CaBPs. Ribbon diagram of CaBPs. Ribbons are shown in gray, EF-hand motifs are in yellow, Ca²⁺ are shown in red (figure generated using GRASP, and aa numbers are for the short form of CaBP1 (see “Materials and Methods”)). Three elements that are different from CaM are marked with arrows; these include the N-terminal myristolylation (CaBP1 and CaBP2), inactivation of EFII-hand motif (all CaBPs), and extra α-helical turn (all CaBPs).

Primary structure of CaBPs and phylogenetic tree. A, sequence alignment of h-CaBP1, h-CaBP2, h-CaBP3, h-CaBP4, h-CaBP5, and h-CaM. Alignment of the deduced aa sequences of h-CaBPs with CaM. The identical residues in all sequences are shown in white letters on black background. The conservative substitutions (E=D; S=T; V=M=I=L; K=R) are shown in white letters on dark gray background. The residues that are identical among CaBPs are shaded in light gray. Functional EF-hand motifs are shown in shaded boxes, and the nonfunctional EF-hand motif is in an open box. Theasterisk represents the myristoylation site, and the down arrowhead over a solid bar indicates a 4-amino acid-long extension of the central a-helix. Arrows indicate the intron/exon junctions for the h-CaBP1, 2, 4, 5, and CaM genes. Vertical bars indicate the intron/exon junctions in CaBP3 and in alternative splice forms of CaBP1 and CaBP2. L- indicates the long spliced forms of CaBPs, and the underlined aa residues are absent in the short spliced forms. The letters h and s above the sequences indicate α-helices and β-strands, respectively. B, phylogenetic tree. The tree was built with a bootstrap analysis of neighbor-joining distance using PAUPSearch in GCG (Genetics Computer Group). The sequences included, with their GenBank™/EMBL accession numbers in parentheses, are: h-CaM (A31920); h-CaM-like protein (P27482); rat caldendrin (Y17048); human recoverin (S62028); GCAP1 (L36859); GCAP2 (see Ref. 30); GCAP3 (AF110002); chicken visinin (P22728); bovine neurocalcin (JH0616); VILIP1: human visinin-like protein 1 (U14747); VILIP2: rat visinin-like protein 2 (P35332); VILIP3: human visinin-like protein 3 (P37235); NCS1: rat neuronal Ca²⁺ sensor 1 (P36610); CaBP4 (unpublished sequence); CaBP1, CaBP2, CaBP3, and CaBP5 are novel sequences reported in this study.

Tissue distribution of CaBP mRNAs. A–C, Northern blot analysis of CaBP mRNAs. Poly(A)⁺ RNA from various human tissues (CLONTECH), from bovine (A and C), and from human retinas (B) were probed with a ³²P-labeled human CaBP1, CaBP2, or CaBP3 cDNA. Control hybridization with a ³²P-labeled G3PDH probe is shown at the bottom. The migration of molecular size standards (in kilobases) are shown on both sides. D–F, PCR analysis of CaBPs transcripts distribution in various mouse tissues. Reverse-transcribed total RNA from multiple mouse tissues was used to amplify by PCR the CaBP1 full coding sequence of CaBP1 and CaBP2 or a partial CaBP5. A positive control was carried with G3PDH-specific primers (data not shown)

Subcellular localization of GFP fusion proteins in transfected cells. Confocal fluorescence of CHO cells transiently transfected with a vector (A), GFP (B), Sh-CaBP1-GFP fusion (C), or Lh-CaBP1-GFP fusion (D).

Myristoylation of CaBPs in bacteria co-expressing human N-terminal myristoyltransferase. A, the expression of CaBPs were done in 30-ml cultures in the presence of 0.7 mCi of [³H]myristic acid. The proteins were affinity purified and separated by SDS-PAGE. CaBPs were identified by Western blot, the band was cut out of the gel, and the radioactivity was measured. CaBP2 was expressed as the short form, and Met⁶ indicated a construct of CaBP5 with the second ATG as the initiation Met. The black line represents the basal activity obtained from similar areas on the gel but without proteins. Similar results were obtained from two independent cultures. B, HPLC purification of CaBP1. CaBP1 was expressed in E. coli, purified on the phenyl-Sepharose column (see “Materials and Methods”), and injected onto a W4 Porex C4 column (Phenomenex). Protein was eluted with 20–100% CH₃CN during 50 min in the presence of 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. The protein peak was analyzed by electrospray mass spectrometry. The predicted (theor) molecular mass was in a good agreement with the observed value, suggesting that CaBP1 was myristoylated.

Inhibition of GRK5 activity by CaBP1. Purified GRK5 (0.8 pmol) were used to phosphorylate rod outer segment membranes (2 μm rhodopsin) in the presence of indicated concentrations of CaBPs. Proteins were separated on a 10% SDS-polyacrylamide gel and visualized by autoradiography. ³²P incorporation into proteins was determined by excising and counting the radioactive bands. The activity of GRK5 in the presence of CaBPs is expressed as a percentage of the rhodopsin phosphorylation in the absence of CaBPs. The short forms CaBP1 and CaBP2 were used in the assays.

Diagram of CaBPs characterized in this study. CaBP1 and CaBP2 are expressed in two spliced forms with an ∼60-amino acid-long insert on the front of the first EF1-hand motif. The inserts are shown as black boxes. This insert sequence has 35% similarity between these two proteins. CaBP1 and CaBP2 contain a consensus sequence for myristorylation. In bacteria, they are substrates for human N-myristoyltransferase (indicated by a circle). CaM has all four EF-hand motifs functional in Ca²⁺ coordination, whereas all CaBPs have nonfunctional EF2-hand motif (open box), either because of deletion of 3 aa (CaBP2) or disabling mutations (CaBP1 and CaBP5). CaBP3 has dissimilar sequences with CaM and other CaBPs through the N-terminal region, EF-hand motifs 1 and 2. All CaBPs are dissimilar in sequence with CaM and other CaBPs through the N-terminal region, EF-hand motifs 1 and 2. The sequences of the C-terminal halves of all h-CaBPs are highly conserved (80%).