Sequence Homology of the Ca2+-dependent Regulator of Cyclic Nucleotide Phosphodiesterase from Rat Testis with Other Ca2+-binding Proteins*

SUMMARY A Ca2+-dependent regulator protein of cyclic 3’:5’-nucleo-tide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca’+-dependent regulator protein is also structurally similar to troponin-C, the CaY+-binding component of muscle troponin and Ca2+ mediator of muscle contrac-tion. The present report describes a partial amino acid sequence of the Cat+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle tro-ponin-C, but is 11 residues shorter than the muscle protein. The Ca’+-dependent regulator protein has an NH,-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of e-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca*+-binding proteins. These proper-. ties may account for the biological specificities demon-strated by these

From the Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030 SUMMARY A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+binding protein with a molecular weight of approximately 17,000. The Ca'+-dependent regulator protein is also structurally similar to troponin-C, the CaY+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Cat+-dependent regulator.
The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein.
The Ca'+-dependent regulator protein has an NH,-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of e-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca*+-binding proteins. These proper-. ties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calciumbinding proteins, our data support the view that all of these molecules contain common sequences, especially at their proposed metal-binding sites.
The cellular role of Ca*+ 1s best understood regarding its regulation of muscle contraction. The Ca2+-binding subunit component of the myofibril is troponin-C (TnC),' which when complexed with troponin-I and -T, regulates the interaction of actin and the myosin cross-bridges in response to changes in the intracellular of other cellular events, such as motility, secretion, division, and metabolic activity (l-6). Non-muscle cells contain a low molecular weight protein (calcium-dependent regulator) which binds Ca2+ with high affinity. Originally identified as a cyclic nucleotide phosphodiesterase activator protein (7, 8), the regulator has subsequently been shown to be structurally similar to skeletal muscle troponin-C (9-13). The amino acid sequences of rabbit (14) and chicken (15) skeletal muscle and bovine cardiac troponin-C (16) are known. Based on a compasison of the sequence homologies of troponin-C to carp parvalbumin, a Caz+-binding protein with a known three-dimensional structure (171, a number of investigators (18-20) have predicted the location of the four sites which bind Ca2+ in troponin-C.
In the present communication, we report? 3 on the partial amino acid sequence of rat testis regulator protein and have compared it with skeletal muscle troponin-C.
Although the rat regulator has physicochemical properties which are similar to troponin-C (13), there are differences in the two proteins.
For example, the regulator has 1 residue of Etrimethyllysine while skeletal troponin-C has a histidine at this position (21). In addition, the regulator has four equivalent Caz+-specific binding sites, whereas two of the four sites in troponin-C also bind Mgz+ (22). Finally, the molecular weight of the regulator is smaller than that of troponin-C by about 1000 (12, 13). The purpose of the present study was to determine the primary structure of the regulator and to relate this information with structure-function properties of the regulator and other calcium-binding proteins.

RESULTS" 3
Isolation of Tryptic Peptides-Regulator (2 pmol) was treated with trypsin and tryptic peptides were fractionated on a column of Sephadex G-50 (Fig. 1). Four major zones of peptide were detected. Rechromatography of Zone A over the same column gave T-5. Acidification of Zone B to pH 1.6 precipitated peptide T-11. The acid-soluble peptides in Zone B were subjected to ion exchange chromatography to yield T-10 and a tryptic peptide consisting of T-2 + T-3; cleavage at the lysine residue in position 21 was only partial. Acidification of Zone C to pH 1.6 yielded an insoluble peptide, T-l. Ion exchange chromatography of the acid-soluble peptides of Zone C gave peptides T-2, T-3, T-4, T-7, and T-9. High voltage * The "Experimental Procedures," Tables I and II Sequence Homologies in Ca2+-binding Proteins electrophoresis of Zone D gave peptides T-6 and T-8. The amino acid composition of each tryptic peptide is shown in Table I.
Isolation of Cyanogen Bromide Fragments -Amino acid analysis of the CNBr digest of regulator without acid hydrolysis showed the presence of both free homoserine and homoserine lactone, indicating a Met-Met sequence. Chromatography of the CNBr digest is shown in Fig. 2; six zones of peptides were pooled. By amino acid analysis, Zone I contained methionine and probably represents uncleaved material. Zone II contained three peptides, CNBr VI-VIII (Fig. 3), which were not further fractionated. l&chromatography of Zone III on Sephadex G-50 yielded a fragment which did not react in the Edman degradation, indicating that it represented the NH&erminal CNBr fragment of the regulator. High voltage paper electrophoresis at pH 3.7 of Zone IV gave CNBr-II and -111. CNBr-V and -X were obtained by paper chromatography of Zone V. CNBr-X was the only fragment which did not contain homoserine and represents the COOHterminal peptide of regulator. Zone VI contained free homoserine. The amino acid compositions of each of the purified CNBr fragments are given in Table II.
Sequence of Regulator-The amino acid sequence of the tryptic peptides was determined by subtractive Edman degradations (25). Some of the peptides were degraded further with chymotrypsin and thermolysin, the derived peptides were isolated, and their sequences were determined (Fig. 3). Peptide T-l was the only tryptic peptide resistant to Edman degradation and thus represents the NH,-terminal peptide. Treatment of T-l with thermolysin yielded four peptides (Fig.  3). Th-1 was ninhydrin-negative and did not react in the Edman degradation.
The alignment of T-2 and T-3 was based on the chymotryptic peptides from a tryptic peptide which contained T-2 and T-3; no cleavage occurred at lysine in position 21. CNBr-II was the only CNBr fragment with NH,-terminal arginine. Thus, an overlap between CNBr-I and -11 was established. The alignment of CNBr-II and -111 was based on the composition of T-5 and on automated Edman degradation at T-5. The sequence of CNBr-III was established from subtractive Edman degradations of the fragment and from its four thermolytic peptides (Fig. 3). The alignment of CNBr-III, -IV, and -V was based on the sequence of T-5 and the fact that T-6 was the only tryptic peptide with NH,terminal methionine and homology to troponin-C. The positioning of T-6 penultimate to T-7 was based on the isolation and sequence of a tryptic peptide designated CNBr-VI-T-1 in Fig. 3 which was isolated from a tryptic digest of a mixture of CNBr-VI, -VII, and -VIII (Zone II, Fig. 2). This peptide was the only peptide to have NH,-terminal lysine and, thus, represents the COOH-terminal part of CNBr-V. Chymotryptic digestion of T-9 yielded C-l and C-2 (Fig. 3). T-10 contained an unknown amino acid. The identification of this residue as l -trimethyllysine has recently been described (21). The thermolytic peptides of T-10 are shown in Fig. 3. The alignment of T-9 and T-10 was based on homology to troponin-C.

DISCUSSION
The amino acid sequence of rat testis regulator was aligned with that of rabbit skeletal muscle troponin-C as shown in Fig. 4. The two proteins are clearly similar in that approximately 50% of the residues represent homologous sequences. Many of the substitutions, in fact, are conservative replacements. Regulator, however, is 7 residues shorter than troponin-C at the NH, terminus and a single residue shorter at the COOH terminus. Based on sequence homology to troponin-C, there is a deletion of 3 residues at positions 85-87. Overall, these changes leave the regulator 11 amino acids smaller than troponin-C, which supports the M, = 1000 difference reported previously by our laboratory (12,13). Regulator also contains, at position 115, an unusual basic amino acid, identified as e-trimethyllysine (21); the homologous position in troponin-C represents the sole histidine residue. The physiological importance for the presence of this rare amino acid is unknown.
These differences in primary sequence may reflect the marked differences in biological, structural, and metal-binding properties reported earlier by our laboratory (12, 13). Each protein is a poor substitute for the other in regulating cyclic nucleotide phosphodiesterase or actomyosin ATPase (12). They also have distinct differences in their structural conformation as determined by circular dichroism and tyrosine fluorescence (13). In addition, troponin-C contains three classes of metal-binding sites (22), while testis regulator contains four equivalent Ca'+-specific sites (13).
Based on the homology between parvalbumin and troponin-C, Kretsinger and Barry (28) have constructed a model of the predicted three-dimensional structure of troponin-C, including the four metal-binding sites. Again, due to sequence homology, the Ca"+-binding sites of testis regulator can be predicted (18). Compiled in Fig. 5 are the metal-binding sites of various Ca2+-binding proteins. The X, Y, and Z notation represents the octahedral binding coordinates located in the EF helical loop required for high affinity metal binding (28). The most outstanding features are the aspartate located at the X and Y coordinates and glutamate at the -2 coordinate. In addition, glycine is invariant between the Y, 2 and the 2, -Y calcium coordinate residues (Fig. 5). This latter characteristic appears to be unique for the lower affinity, Ca"+-specific sites (18). The exception to these observations is bovine troponin-C site 1, which has leucine and alanine, respectively, at the X, Y positions and glutamate replacing the invariant glycine between the Y, Z positions. As proposed by vanEerd and The Ca2+-dependent regulator has been found in essentially all eukaryotic cells examined and is in considerable excess compared to that necessary to regulate cyclic nucleotide phosphodiesterase (29, 30). The protein is furthermore found in high quantities in secretory tissues, including adrenal medulla, testis, platelets, and brain (13,29,(31)(32)(33). It has also been shown to regulate, via Ca2+, several enzyme systems including phosphodiesterase (34, 351, adenylate cyclase (36, 37), skeletal muscle actomyosin ATPase (12), and recently erythrocyte membrane Ca *+-ATPase (38,391. Wang and Desai (40) have isolated an additional unidentified protein which binds regulator in a Caz+-dependent manner. Calcium is well known to be involved in regulating cellular motility, secretion, division, and metabolic activity (l-6). It has been estimated that the Ca2+ levels increase from 1Om8 to lo-" M during cell activation (4). Regulator (KdcaP+ -10-O M) also displays significant conformational changes and subsequent phosphodiesterase activation within these calcium concentrations (13). In addition, using monospecific antibody to testis regulator, our laboratory has localized this protein within cells by indirect immunofluorescence.
Regulator fluorescence is associated with filamentous cables in the cytoplasm, the mitotic half-spindle of metaphase and anaphase, and the midbody of late telophase.4 Collectively, these observations suggest that the regulator plays a fundamental cellular role as a Ca'+ receptor or mediator of Ca2+-stimulated events.