Isolation and Molecular Characterization of AKAP110, a Novel, Sperm-Specific Protein Kinase A-Anchoring Protein

Srinivasan Vijayaraghavan, Greg A. Liberty, Jag Mohan, Virginia P. Winfrey, Gary E. Olson and Daniel W. Carr

Kent State University (S.V., J.M.) Kent, Ohio 44242
Veterans Affairs Medical Center and Oregon Health Sciences University (G.A.L., D.W.C.) Portland, Oregon 97201
Department of Cell Biology (V.P.W., G.E.O.) Vanderbilt University Nashville, Tennessee 37232


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
cAMP-dependent protein kinase A (PKA) is a ubiquitous, multifunctional enzyme involved in the regulation of a diverse array of cellular events. PKA holoenzyme consists of four subunits, two catalytic and two regulatory. In the absence of cAMP, the regulatory (R) subunits keep the catalytic subunits inactive. cAMP binding to the R subunit promotes dissociation and activation of the catalytic subunit. Recent studies have shown that PKA is anchored at specific subcellular sites through the interaction of the R subunit with A-kinase anchoring proteins (AKAPs) (1, 2, 3, 4). A number of AKAPs have been cloned and biochemically characterized (2). All AKAPs contain a common structural motif that binds with nanomolar affinity to the R subunit of PKA (5, 6). Other anchoring proteins are capable of binding both RI and RII and have been labeled dual D-AKAPs (7, 8). Since PKA has broad substrate specificity, presumably one of the primary functions of PKA anchoring is to spatially restrict its action, thus ensuring specificity of function. In addition to PKA, some AKAPs also simultaneously bind other signal transduction molecules such as calmodulin (9), calcineurin (10), and protein kinase C (11, 12). Thus, AKAPs could serve as scaffolding proteins that coordinate the actions of several signal transduction enzymes.

cAMP analogs and agents that increase intracellular cAMP are potent stimulators of sperm motility (13, 14). The highly polarized sperm cell is particularly well suited for investigating the structure and function of PKA- anchoring proteins. Differentiated sperm have a number of distinct subcellular structures. The microtubular apparatus in mammalian sperm is surrounded by distinct cellular organelles such as the outer dense fibers and fibrous sheath. Free diffusion of plasma membrane proteins is apparently restricted to domains defined by the sperm head, midpiece, and tail regions (15). Similar restrictions to the free diffusion of the cytosolic contents may exist. The cytoplasmic volume of the sperm cell is considerably lower than that of most somatic cells. This unique compartmentalization of the sperm cell make it an excellent model system by which to study the role of targeting and anchoring of PKA and other enzymes in regulating cell function.

We have recently shown that membrane-permeable peptides, designed to disrupt RII interaction with AKAPs, cause the arrest of sperm motility (16). These data suggest that PKA anchoring is essential for sperm motility. Sperm contain both RI and RII isoforms of the regulatory subunit (17). Immunocytochemical analysis shows that RI{alpha} and RIß subunits are predominantly localized in the acrosomal segment of the head whereas RII{alpha} is confined to the sperm flagellum. This differential localization implies distinct roles for the type I and type II PKA isoforms in sperm functions. We have also shown that bovine, human, monkey, and mouse sperm all contain one predominant AKAP with a relative mol wt of approximately 110,000 (AKAP110) (16). The objective of this report is to determine the structure of AKAP110 by molecular cloning, ascertain its biochemical properties, and document its subcellular localization within sperm.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Purification and Microsequencing of AKAP110
AKAP110 was purified from bovine caudal epididymal sperm. AKAP110 was very insoluble, even after incubation with high concentrations of salt and detergent. Therefore, the purification procedure consisted first of washing away all soluble proteins using homo-genization buffer containing high salt and detergent (see Materials and Methods). In the final step, the AKAP110- containing pellet was solubilized with 9 M urea, and the proteins were separated by two-dimensional electrophoresis. A spot corresponding to AKAP110 (detected by RII overlay assay on a parallel gel (Fig. 1Go)) was cut out and sent to Harvard Microchemistry for tryptic digestion, separation on HPLC, and amino acid sequencing. Seven different peptide fragments were sequenced. Comparison of these sequences with the GenBank databases revealed no sequence identity with any known protein, suggesting this was a novel sperm protein. However, several of these sequences did show sequence similarity to expressed sequence tags (ESTs), which had been isolated from both human and murine testis libraries (MTEST21, AA424242, AA492960 and AA061683).



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Figure 1. Purification of AKAP110 from Bovine Sperm

An RII overlay assay was performed on bovine sperm extracts that were prepared as described in Materials and Methods. Sperm extracts were subjected to either one-dimensional (left side of blot) or two-dimensional electrophoresis. In the first dimension, isoelectric focusing was performed over a pH range of 3.0–10.0. The large arrow indicates the direction of isoelectric focusing from basic to acidic. The small arrow points to a spot on the two-dimensional gel that was excised and used for amino acid sequencing.

 
Cloning and Sequence Analysis of cDNA-Encoding Sperm AKAP110
Two of the murine ESTs, AA061683 and AA492960, were obtained from Research Genetics, Inc. (Huntsville, AL) and used as probes to screen a mouse {lambda} zap testis library (Stratagene, La Jolla, CA). After the probe was radiolabeled, three positive clones were plaque purified from approximately 100,000 recombinants. The inserts were isolated by PCR amplification using primers complementary to the vector on either side of the insert. Two of the inserts were approximately 2.5 kb in length while the third was 3.1 kb. Sequence analysis revealed that the longer clone contained the same sequence as the shorter clones but with 600 additional bases on the 5'-end. The complete DNA sequence for murine AKAP110 has been deposited in GenBank (see footnote 1). The sequence surrounding an initiation Met codon at nucleotides 289–294 (AGGATGG) meets all the requirements for a consensus start site (18, 19). The 5'-upstream sequence, nucleotides 1–288, contains 10 inframe stop sites, precluding the possible use of another ATG upstream. A termination site is found at nucleotides 2884–2886 and is followed by a poly-A tail starting at base 3079.

The open reading frame between bases 289 and 2886 predicts that AKAP110 contains 864 amino acids with a calculated mol wt of 95,578. A comparison of the amino acid sequence of AKAP110 with the Prosite database (20) of patterns produced no matches, except for patterns with a high probability of occurrence such as phosphorylation sites. Analysis of the sequence using the SOSUI computer program (21) predicts that AKAP110 is a soluble as opposed to a membrane protein.

Comparison of Human, Murine, and Bovine AKAP110
As most of our work had been done on bovine sperm and the AKAP110 had been isolated from bovine sperm, we wanted to characterize the bovine homolog of AKAP110. The murine cDNA was radiolabeled and successfully used as a probe to screen a bovine testis library. To explore the potential of this protein as a contraceptive target, we also needed to obtain the human sequence. This was achieved by using a 5'-RACE (rapid amplification of cDNA ends) protocol. The gene-specific primer was designed using the human EST sequence (GenBank accession no. AA424242, unpublished). An alignment of the human, murine, and bovine sequences are shown in Fig. 2Go. The sequences for AKAP110 from all three species have been submitted to GenBank.1 All seven of the peptide sequences obtained from the Harvard Microsequencing Facility aligned perfectly within the bovine AKAP110 sequence (underlined sequence in Fig. 2Go), confirming that the cloned protein is an identical match with the biochemically isolated protein. Approximately 65% (554 of the 864) of the amino acids are conserved (identical) in all three species. Some regions are much more conserved than others, e.g. the last 200 amino acids at the C terminus are highly conserved (160 of 200 identical for all three species), suggesting these regions contain homologous domains that may have important functional roles in sperm.



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Figure 2. Alignment of Derived Amino Acid Sequences of AKAP110 from Human, Murine, and Bovine

The alignment of AKAP110 from three mammalian species was performed using MacVector software (Oxford Molecular Group). Identical amino acids are boxed and shaded. Both the cDNA and amino acid sequence from all three species have been deposited in the GenBank database.

 
Production of Antibodies against AKAP110
One of the seven peptide sequences obtained from Microsequencing of the bovine sperm protein (SCVETLGEHIIK) was synthesized and used to produce antisera from rabbits (see Materials and Methods). Polyclonal antibodies against AKAP110 were affinity purified using the synthetic peptide attached to an Amino Link (Pierce Chemical Co., Madison, WI) matrix. To test the antibodies, full-length AKAP110 was subcloned into a pET30a vector, and expression was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) (Fig. 3Go). IPTG-induced full-length AKAP110 was detected using both an RII overlay assay and a Western blot analysis with the AKAP110 peptide antibody. The antibodies also recognized a 110-kDa protein in bovine sperm extracts (Fig. 3CGo). The observation that the expressed protein migrates at approximately the same Mr as the native sperm protein suggests that the native protein is not processed or cleaved within the cell. These antibodies were used for immunofluorescent studies described later in this manuscript.



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Figure 3. Characterization of Antibodies against AKAP110

RII overlay and Western blots were prepared as described in Materials and Methods. The blots were probed with either radiolabeled RII{alpha} (A) or affinity- purified anti-AKAP110 antibody (dilution 1:5,000) (B and C). Panels A and B are identical blots loaded with bacterial lysates from either uninduced (Control) or IPTG-induced full-length AKAP110, which has been separated into supernatant (Supe) and pellet fractions. The blot in panel C received 50 µg of bovine caudal sperm lysate.

 
Identification of the RII-Binding Domain
Truncated fragments of murine AKAP110 were subcloned into a pET30a vector, expressed in Escherichia coli, and used to determine the RII-binding site on AKAP110. Expression of protein fragments was monitored by Western blotting analysis, while the binding of AKAP110 fragments to RII was monitored using the RII overlay assay (Fig. 4Go, A and B). Deletion of the C-terminal half of the protein (amino acids 471–864) had no effect on RII binding, while removal of the N-terminal half (amino acids 1–470) totally abolished detectable RII binding. By progressively shortening the N-terminal half, the binding domain was narrowed down to the region between amino acids 117 and 164.



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Figure 4. Identification of the RII-Binding Domain

Full-length AKAP110 (1–864) or truncated fragments 1–470, 471–864, 1–350, 1–164, and 1–117 were expressed in E. coli, separated by electrophoresis, and transferred to Immobilon as described in Materials and Methods. The blots were probed with either radiolabeled RII{alpha} (A) or horseradish peroxidase-conjugated S-Protein, which detects a S-tag that is expressed as a fusion protein by the pET30a vector (B). An RII overlay analysis of bovine sperm was performed either in the absence (panel C, lane 1) or presence (panel C, lane 2) of 1 µM AKAP110 (127–140) peptide (FYANRLTNLVIAMA).

 
To identify the precise amino acids involved in binding RII, we first performed a computer-assisted alignment (MacVector, Oxford Molecular Group, Campbell, CA) of other AKAP RII-binding domains that had previously been identified and then had the computer align all these other sequences with AKAP110. The best alignment occurs with a stretch of amino acids between 124 through 141. This region is 100% conserved in all three species. To confirm that this region was sufficient for binding RII, a synthetic peptide (AKAP110 127–140, FYANRLTNLVIAMA) was added to an RII overlay assay. This peptide (1 µM) effectively blocked RII binding to AKAP110 when used as an antagonist in the overlay assay (Fig. 4CGo).

We have previously predicted that an amphipathic helical secondary structure was necessary for AKAP binding to RII, but a high degree of variability was apparently allowed at each position in the primary structure (5). Now that more RII-binding domains have been identified, we are able to refine our definition of this motif. Optimal alignment of 15 putative AKAP- binding domains was performed using the MacVector ClustalW program (Fig. 5AGo). This analysis revealed that 8 of 18 positions were strongly conserved with only a limited number of amino acid substitutions allowed. All the conserved positions contained mostly hydrophobic amino acids. A common motif is emerging and can be represented as X{L,I,V}X3{A,S}X2{L,I,V}{L,I,V}X2{L,I,V}{L,I,V}X2{A,S}{L,I,V}, where X represents any amino acid and single letters in the braces represent conserved amino acids that are favored at that position. Eleven of the 15 AKAPs match at least 7 of the 8 conserved positions. If we allow fairly conservative substitutions (V for A,S at positions 6 and 17) and (T, A, or M for I,L,V at positions 2, 10, 13, 14 and 18), then 9 of the 15 AKAPs are conserved at all 8 positions and 13 of 15 are conserved at 7 of 8 positions. Six of the 8 conserved positions consist of branched aliphatic amino acids that are most likely involved in the binding to hydrophobic amino acids on the N terminus of RII. The other two positions, 5 and 17, are occupied mostly by A and S, which are residues with low steric bulk, either amphiphilic or hydrophilic neutral and ambivalent in terms of structural position, i.e. they can be located either on the internal or external face of the protein. These amino acids likely fit into size-restricted pockets on RII. Positions 2 and 18 contain mostly hydrophobic residues (L,I,V), but there seems to be more wobble, or nonconservative substitutions, allowed toward the ends of the binding domain than in the central core. When drawn as an {alpha}-helix, all of the conserved positions align to one face on the {alpha}-helix (Fig. 5BGo). The amino acids that occupy positions between the conserved residues (positions 3, 4, 7, 8, 11, 12, and 15) are more variable, although certain preferences are obvious. Amino acids at these positions are mostly (~90%) hydrophilic or neutral residues. These data are consistent with a model of the AKAP/RII binding domain containing an amphipathic helix with a highly conserved hydrophobic face and a less conserved hydrophilic face. The binding domain appears to encompass five turns of an {alpha}-helix. Work is currently in progress to determine the relative contribution of each conserved position to the AKAP/RII binding affinity. We would predict that the affinity between any particular AKAP and RII would be related to how closely the AKAP-binding domain matches the conserved motif, especially the positions in the middle of the domain (6, 9, 10, 13, 14, 17). AKAP110 is conserved at all six of these central positions (Fig. 5CGo).



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Figure 5. Alignment and Helical Wheel Projection of the RII-Binding Domains from 15 Known AKAPs

Optimal alignment of the RII-binding domains was performed with the assistance of the MacVector ClustalW program (A). The name of the AKAP and the sequence numbers corresponding to the RII-binding domain are listed on the left. The shaded areas represent groups of amino acids with conserved functional properties. Helical wheel representation of the eight conserved positions is presented in panel B. The letter X indicates a nonconserved position. The letters in the top row at positions 2, 6, 9, 10, 13, 14, 17, and 18 indicate the conserved amino acids favored at this position, and the letters in the bottom row are conservative substitutions that appear to be allowed at this position. The helical wheel representation of the RII-binding domain of AKAP110 is shown in panel C.

 
Comparison of AKAP110 with AKAP82
A comparison of the sequence of AKAP110 with nonredundant GenBank sequences using the Gapped BLAST program (22) revealed that AKAP110 contained significant sequence homology (35% identities under optimal alignment, 20 gaps) with a sperm major fibrous sheath protein (23, 24, 25) also known as p82, AKAP82, FSC-1, and HI. The sequence similarity exists over the entire length of the molecule, although the longest stretch of identical amino acids is only 11. Several segments of sequence are quite conserved (defined as a stretch of at least 20 amino acids with >50% identity) (Fig. 6Go), suggesting that these two divergent proteins still contain similar domains and may still perform similar function. Conversely, more than half of the sequence is not similar, suggesting each protein may have evolved unique functions. It is interesting to note that there are two domains (domains 1 and 2 in Fig. 6Go) in the N terminus that are conserved, even though these domains are cleaved off of the processed FSC/AKAP82 protein. Domain 3 contains the RII-binding site for both proteins. Comparison of the 18 positions within the RII-binding domain (see Fig. 5AGo) reveals that only 12 of the 18 residues are identical.



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Figure 6. Comparison of Common Domains between AKAP110 and AKAP82

Common domains between AKAP110 and FCS/AKAP82 are defined as segments of sequence that are at least 20 amino acids long and contain at least 50% sequence identity. These domains are represented by solid boxes. Both proteins are drawn from the N terminus to the C terminus with the amino acid numbers indicated by the scale at the bottom.

 
Tissue Distribution of AKAP110 mRNA
To determine the tissue distribution of AKAP110, Northern blots containing 2 µg of poly(A+) RNA from different adult mouse tissues were probed with 32P-labeled full-length AKAP110 cDNA. A single message was detected only in the testis (Fig. 7Go), suggesting that this protein is sperm specific. This conclusion is supported by the finding that ESTs with similar sequences have been isolated only from testis libraries. The probe bound to a message of approximately 3.6 kb. To ensure the probe was not hybridizing with the mRNA from the major fibrous sheath protein, a segment of AKAP110 cDNA (bases 838 to 1053), which contains no significant sequence identity with the fibrous sheath cDNA, was radiolabeled and used as a probe on the same blot. This shorter, AKAP110-specific probe also bound to a 3.6-kb message (data not shown), indicating that there was no cross-hybridization between the two messages.



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Figure 7. Northern Analysis of AKAP110 mRNA in Eight Murine Tissues

A Northern blot containing 2 µg of poly(A+) RNA per lane was probed with 32P-labeled AKAP110 cDNA and detected by autoradiogram. The source of the poly(A+) RNA is indicated at the top of each lane, and the Mr (bp x 10-3) is indicated on the left.

 
In Situ Hybridization of AKAP110 in Testis
Antisense AKAP110 riboprobe specifically hybridized to the seminiferous epithelium, and no detectable signals were noted either in the interstitial tissue or in the peritubular and Sertoli cells of the seminiferous tubules (Fig. 8AGo). AKAP110 exhibited a stage-specific expression pattern in germ cells. No hybridization signal was detected in spermatogonia or spermatocytes, but expression was evident in round spermatids (Fig. 8BGo). Late spermatids also exhibited no detectable expression of AKAP110 (Fig. 8BGo). AKAP110 sense riboprobes gave no detectable signals (Fig. 8CGo).



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Figure 8. In Situ Hybridization of AKAP110 in Testis

A, In situ hybridization of mouse testis showing pattern of labeling with AKAP110 antisense probe in several seminiferous tubules. Hybridization is specific to the round spermatids (RS) of the seminiferous epithelium. Dotted lines represent boundary between adjacent seminiferous tubules. L, Tubule lumen. B, Higher power view of single seminiferous tubule hybridized with antisense AKAP110 probe. The bracket represents the region of the seminiferous epithelium that includes spermatogonia, spermatocytes, and the basal aspect of the Sertoli cells; note that no positive signal is detected in this region. In contrast, the immediately superficial layer of RS exhibits a strong hybridization signal. Late spermatids (LS) adjacent to the tubule lumen (L) do not exhibit detectable signal. C, In situ hybridization of mouse testis probed with AKAP110 sense probe. No specific hybridization is noted in any cell type including the round spermatids (RS). The dotted line represents the junction of two adjacent seminiferous tubules. L, Tubule lumen.

 
Subcellular Localization of AKAP110
Ejaculated and cauda epididymal spermatozoa exhibited identical specific staining patterns with anti-AKAP110 antibody; no differences in staining were noted if spermatozoa were permeabilized before or after formaldehyde fixation. Spermatozoa immunostained with anti-AKAP110 exhibited intense fluorescence of the anterior acrosomal segment, but no staining was noted over the equatorial or postacrosomal segments of the head. Detached acrosomal caps also exhibited an intense fluorescence, and no fluorescence was noted on heads after release of the acrosomal cap (Fig. 9Go, A and A'). Spermatozoa immunostained with anti-AKAP110 exhibited specific fluorescence along the entire flagellum (Fig. 9Go, A and A'). No fluorescence was detectable in spermatozoa stained with identical dilutions of preimmune serum (Fig. 9Go, B and B').



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Figure 9. Subcellular Localization of AKAP110 in Bovine Spermatozoa

Matched phase contrast (A and B) and fluorescence (A' and B') of cauda epididymal spermatozoa immunostained with anti-AKAP 110 (A and A') or preimmune (B and B') serum. The spermatozoa shown were permeabilized by Triton X-100 extraction before formaldehyde fixation. Spermatozoa immunostained with anti-AKAP 110 (A and A') exhibit intense staining of the acrosomal segment (AC) and an absence of detectable staining over the posterior head domain. Acrosomal caps released from the sperm head (AC*) retain an intense fluorescence, while heads without an adherent acrosomal cap are devoid of detectable staining (H). The flagellum (F) also exhibited specific staining. Spermatozoa stained with preimmune serum (B and B') exhibited no fluorescence. All photos are at the same magnification and the bar in panel A is equal to 15 µm.

 
DISCUSSION
A substantial portion of mammalian sperm PKA is insoluble and is presumed anchored to AKAPs. A single predominant RII-binding protein (Mr of ~120,000) in bovine sperm was first identified by George Orr and colleagues in 1984 (26). A similar sized RII-binding protein has since been detect in other mammalian species including mouse (27), rhesus monkey (16), and human (16). This protein migrates at approximately 110 kDa in SDS-PAGE and is thus designated AKAP110. We have now isolated, cloned, and characterized AKAP110 from mouse, bovine, and human sperm. Several lines of evidence confirm that the AKAP110 cDNA clones correspond to the biochemically isolated sperm AKAP110 protein: 1) All seven peptide sequences obtained from microsequencing of the bovine sperm AKAP110 protein are present in the open reading frame of the bovine AKAP110 cDNA; 2) The recombinant protein expressed in bacteria binds RII (Figs. 3Go and 4Go); and 3) Antibodies against a synthetic polypeptide within AKAP110 recognize both the recombinant and native AKAP110 (Fig. 3Go).

AKAP110 appears to be expressed only in spermatids and spermatozoa. Use of the full-length AKAP cDNA (3 kb) as a probe in Northern blot analysis and exposure of the blot for extended periods failed to show a detectable message in any tissue except testis (Fig. 7Go). In situ hybridization analysis detected AKAP110 only in round spermatids (Fig. 8Go), suggesting this protein is not expressed in any testis cells other than spermatids. The absence of AKAP110 transcript in spermatogonia and spermatocytes also suggests that AKAP110 is transcribed postmeitically.

The calculated molecular mass of 95 kDa and its isoelectric point (5.9) are in reasonable accordance with the corresponding experimentally observed values (Fig. 1Go). Comparison of the amino acid sequence of sperm AKAP110 from three different species reveals significant homology. There is an overall 65% amino acid sequence identity. There are large domains with greater than 95% homology interspersed by a region of deletions [beginning at amino acid (a.a.) 45] and a region with significant variation in amino sequence (~ a 50-amino acid segment surrounding a.a. 500). The conserved regions presumably correspond to functionally conserved domains. For example, the putative RII-binding domains (a.a. 124–141) are completely conserved in proteins from all three species. Further comparative studies with sperm AKAP110 isolated from other nonmammalian species may be instructive.

The amino acid sequence of AKAP110 has a 35% homology to the major fibrous sheath protein (FSC/AKAP82) (23, 24). We have identified eight regions of high homology (>50% sequence identity) between these two proteins (Fig. 6Go), suggesting these proteins may share common functions. FSC/AKAP82 is synthesized as a larger (97 kDa) precursor protein (28). The amino terminus moiety (a.a. 1–180) of the precursor protein is cleaved during sperm maturation in the testis (28). The FSC/AKAP82 protein in mature sperm is 82 kDa in size. It is interesting to note that two of the domains conserved between these two proteins are in the region that is cleaved off of FSC/AKAP82 but not off AKAP110, suggesting these domains might be vital for the functioning of AKAP110 and might hinder the functioning of FSC/AKAP82. In spite of the significant homology between these two proteins, there was no cross-reactivity between these two proteins either at the DNA or antibody level. The mRNA of AKAP110 is 3.5 kb (Fig. 7Go) compared with 3.0 kb (24) or 3.1 kb (23) reported for FSC/AKAP82. The use of the entire full-length AKAP110 cDNA to probe a Northern blot or a cDNA library resulted in the detection of only the AKAP110 message or AKAP110 cDNA, respectively. Antibodies generated against AKAP110 detect only a single protein band at 110 kDa (Fig. 3Go), but not the unprocessed or processed FSC/AKAP82. The sequence similarities between these two proteins suggest they share a common origin. However, the subcellular location of the two proteins appears to be different, suggesting that the domains that are responsible for determining the location of these proteins are not conserved. The exact evolutionary relationship between these proteins should provide valuable insights into the origin and function of these proteins in mammalian sperm. Particularly interesting will be the determination of the existence of AKAP110-like proteins in sperm from invertebrates, which are cAMP responsive, but lack several of the accessory structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers found in mammalian sperm.

The major fibrous sheath protein, FSC/AKAP82, is reported to be a RII- binding protein (23, 29). However, compared with AKAP110, we can only detect this protein faintly or not at all by RII overlay analysis (see Figs. 1Go and 4CGo, this manuscript, and Fig. 1CGo of Ref. 16). One explanation for this may be that AKAP110 has a higher affinity for RII than FSC/AKAP82. Our analysis of the requirements for RII binding (see Results) would predict that this would be the case. Although there is a significant homology (12 of 18 amino acids are identical) between the putative RII-binding domain of AKAP110 (Fig. 5Go) and the corresponding region in FSC/AKAP82 (29), some differences between these two proteins in this sequence segment occur at conserved positions required for RII binding. Specifically, FSC/AKAP82 has a valine at position 6 instead of alanine or serine and a serine at position 10, where the permitted amino acids are leucine, isoleucine, valine, alanine, and threonine. It is possible that these differences in the core of the binding domain would reduce affinity of FSC/AKAP82 for RII.

The subcellular localization of PKA in sperm is particularly interesting because the RI isoforms are located in the acrosomal region of the head, while RII{alpha} is confined to the flagellum. Immunocytochemical studies show that AKAP110 is present in both the acrosomal region of the head and along the entire length of the flagellum, suggesting AKAP110 might interact with both RI and RII. Dual specificity AKAPs, anchoring proteins that bind both R1 and RII PKA isoforms, have previously been reported (7, 8), but these data present the intriguing possibility that a single AKAP may anchor two different PKA isoforms at two distinct locations within the same cell. Using a yeast two-hybrid system protocol, AKAP82 has recently been shown to bind to RI, and the binding sites have been identified (30). Based on the sequence homology between AKAP110 and AKAP82, we can now reasonably predict that AKAP110 does bind RI. There is significant sequence homology between the RI-binding site (YANQVASDMM) on AKAP82 and a similar site on AKAP110 (YANSVVSDMM). The conservative substitutions (Q to S at position 4 and A to V at position 6) would not disrupt the amphipathic helix nature of the binding domain. These observations support the hypothesis that AKAP110 is a dual AKAP. Studies are in progress to test this hypothesis.

In summary, we have cloned and characterized a novel sperm AKAP from three mammalian species and have identified common domains, including the RII binding domain, between these species and between AKAP110 and other sperm proteins. Since PKA anchoring appears to be a key mechanism regulating sperm motility and possibly other sperm function, AKAP110 might be a logical target for the development of a sperm-based contraceptive, especially as AKAP110 appears to exist only in sperm cells. Identification of the human AKAP110 should also facilitate studies to identify mutations that may be associated with some cases of idiopathic male infertility.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 MATERIALS AND METHODS
 REFERENCES
 
Sperm Extract Preparation
Testes from mature bulls with intact tunica were obtained from a local slaughterhouse, and sperm from caudal epididymis were isolated and washed as previously described (31). The washed sperm were then processed into heads, tails, and midpieces as previously described (31, 32). The tails were pelleted at 16,000 x g for 15 min and the supernatant discarded. The tail pellet was resuspended in homogenization buffer (50 mM Tris-HCl, pH 7.0, 2 mM EGTA, 2 mM EDTA, 0.5 M NaCl, 1% NP-40, 0.1% ß-mercaptoethanol, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 10 µg/ml trypsin inhibitor, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml N{alpha}-tosyl-lys-chloromethyl- ketone) and sonicated at 4 C for 20 sec at setting 4 on a Branson Sonifer 450 (Branson Ultrasonics Corp., Danbury, CT). Suspension was inverted at 4 C for 30 min and then centrifuged at 16,000 x g for 15 min at 4 C. The pellet was resuspended again in homogenization buffer, inverted at 4 C for 30 min, and centrifuged at 16,000 x g for 15 min at 4 C. The remaining insoluble pellet was resuspended in solubilization buffer (deionized 9 M urea, 4% NP-40, 2% ß-mercaptoethanol, and 2% Ampholytes, pH 3–10 (Pharmacia Biotech, Piscataway, NJ) and rocked at room temperature for 2 h. A sample was then used for two-dimensional electrophoresis. For the one-dimensional electrophoresis experiment shown in Fig. 1AGo, the homogenization buffer lacked the 0.5 M NaCl and the 1% NP-40.

Screening of cDNA Libraries
Approximately 0.5 x 106 plaques from a mixed germ cell mouse testis {lambda}ZAPII cDNA library (Stratagene) were screened using a synthetic oligonucleotide probe corresponding to a mouse testis EST sequence that matched one of the peptide sequences obtained from microsequencing of bovine sperm AKAP110. The probe was end labeled with 32PO4. Three positively hybridizing clones were plaque purified. Plasmids with inserts were obtained by in vitro excision of pBlueScript SK-fragment in the {lambda}ZAPII phagemid by following the protocol provided by the manufacturer (Stratagene). One of the plasmids had a cDNA insert that contained the entire coding sequence for AKAP110. The nucleotide sequence of the longest insert (3 kb) is shown in this report. Next we screened a bovine testis library prepared by oligo dT priming of poly A+ RNA isolated from adult bovine testis ({lambda}ZAPII cDNA Cloning Kit, Stratagene). Approximately 0.7 x 106 clones were screened at high stringency with 3 kb randomly labeled cDNA of mouse AKAP 110. Inserts from 12 plaque-purified positive clones were sequenced. The longest clone (2.9 kb) included nucleotide sequences corresponding to all but four of the amino-terminal amino acids of AKAP 110. Further screening of the bovine testis cDNA library failed to yield clones with longer inserts. Finally, we screened a human testis cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) and obtained several hybridizing clones. The longest insert of the positive clone was 1.3 kb, which corresponded to the 3'-end of the gene. The 5'-sequences were obtained by PCR of the 5'-stretch human testis cDNA (CLONTECH Laboratories, Inc.) using gene-specific oligonucleotide primers.

DNA Sequence Analysis
Analysis of sequence data, sequence comparison, and alignments were performed using MacVector ClustalW program (Oxford Molecular Group) and the BLAST program (22) provided by the NCBI server at the National Library of Medicine/NIH.

Expression of Full-Length and Truncated AKAP110
All of the expression studies were performed using the murine AKAP110. A DNA insert that encodes for full-length AKAP110 was created by PCR using a 5'-primer (5'-CCGGAATTCATGGCGGATAGGGTTGACTGG-3') designed to place an EcoRI site immediately in front of the ATG codon and a T7 primer for the 3'-end. Amplified cDNA was digested with EcoRI and XhoI and ligated into the expression plasmid pET30a (Novagen, Madison, WI), which was cleaved with the same restriction endonucleases. Full-length AKAP110 was expressed in E. coli BL21 (DE3). Deletion mutagenesis followed a similar strategy as that used to create full-length AKAP110. Fragments were produced using primers designed to create an EcoRI site on the 5'-end and a SalI site on the 3'-end. Mutants were verified by DNA sequencing. Amplified cDNAs were digested and ligated into pET30a and expressed in E. coli.

Production and Purification of Antibodies Directed against AKAP110
Antibodies were produced using a peptide (SCVETLGEHIIK) corresponding to position 448–459 of bovine AKAP110. The peptide was synthesized, conjugated to keyhole limpet hemocyanin, and injected into two different rabbits. After 6 weeks the rabbits were bled and the serum pooled. The antibodies were then purified by precipitation with 40% ammonium sulfate followed by affinity chromatography using the original peptide conjugated to Amino Link matrix (Pierce Chemical Co.).

Western Blot Analysis
Protein extracts were separated on SDS-PAGE and transferred to Immobilon P (polyvinylidene difluoride) membranes. After treatment with Blotto (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% nonfat milk, and 0.1% BSA) to prevent nonspecific binding, the blot was incubated with primary antibodies at the dilutions indicated. After washing, the blots were incubated with antirabbit secondary antibody conjugated to horseradish peroxidase. A final wash was followed by development with an enhanced chemiluminescence kit (Renaissance, New England Nuclear, Boston, MA).

Assay for RII{alpha}-Binding Activity
Proteins were separated and transferred to Immobilon P using a protocol identical to that described for Western blotting. The blots were then probed with 32P-labeled RII{alpha} (5 x 105 cpm/ml), and RII-binding proteins were visualized by autoradiography as previously described (33, 34).

Northern Gel Analysis
A multiple-tissue RNA blot containing size-fractionated murine poly(A+) RNA (2 µg/lane) was obtained from CLONTECH Laboratories, Inc.. The blot was probed with 32P-labeled cDNA (1.5 x 106 cpm/ml)(Random Heximer Labeling Kit, Amersham, Arlington Heights, IL) corresponding to either the full-length murine AKAP110 or nucleotides 838-1055. Hybridization, washing, and autoradiography were performed as previously described (CLONTECH Laboratories, Inc. manual).

Immunofluorescence Microscopy
Immunostaining was performed on both ejaculated and cauda epididymal bovine spermatozoa, and no differences between the sperm populations were detectable. Ejaculated bovine spermatozoa diluted in an egg yolk-citrate extender (American Breeders Service, De Forest, WI) were stored on ice and used within 24 h of collection. Cauda epididymides were obtained from freshly killed slaughterhouse animals, and the spermatozoa were obtained by retrograde flushing of the tubule with PBS. Ejaculated and cauda epididymal sperm suspensions were centrifuged at 1000 x g for 10 min, and the sperm pellets were washed twice by resuspension in HBBS or PBS followed by centrifugation as above.

Spermatozoa were permeabilized either before or after formaldehyde fixation. For prefixation permeabilization, spermatozoa were incubated at 4 C for 30 min in 0.25% Triton X-100, 10 mM DTT, 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM benzamidine, 1 µg/ml leupeptin, and 1 µg/ml pepstatin and then fixed in 4% formaldehyde in 0.1 M sodium phosphate, pH 7.4. For postfixation permeabilization, spermatozoa were initially fixed for 30 min at 4 C in 4% formaldehyde in 0.1 M sodium phosphate, pH 7.4, and then permeabilized for 30 min by the addition of Triton X-100 to a final concentration of 0.25%. Spermatozoa were then allowed to attach to poly-L-lysine coverglasses. Coverglasses were either incubated in absolute acetone at 20 C for 10 min and air dried or processed directly for immunostaining.

Spermatozoa were rinsed in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20 (TNT) and then incubated for 1 h in a blocking solution composed of TNT containing 5% normal donkey serum and 2.5 BSA. Parallel coverslips were then incubated for at least 1 h in equivalent dilutions of preimmune or anti-AKAP110 serum diluted in blocking solution. After three rinses in TNT containing 1% donkey serum, the coverslips were incubated in Cy3-conjugated, affinity-purified, donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After three rinses in TNT, sperm were observed by fluorescence and phase contrast microscopy.

In Situ Hybridization
Sense and antisense riboprobes were synthesized in the presence of digoxigenin-labeled nucleotides (Boehringer Mannheim, Indianapolis, IN) by run off in vitro transcription of linearized pCRII plasmid (Invitrogen, San Diego, CA) containing a 250-bp AKAP110 insert. Nonisotopic in situ hybridization was performed as described previously (35, 36) using cryosections of fresh-frozen mouse testes that were fixed with 4% formaldehyde in 0.1 M sodium phosphate, pH 7.4, and then acetylated for 15 min with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0. Riboprobes were denatured for 5 min at 80 C, diluted in hybridization buffer composed of 50% formamide, 10% dextran sulfate, 4x SSC, 1x Denhardts reagent, 0.5 mg/ml heat-denatured herring sperm DNA, and 0.25 mg/ml yeast tRNA and hybridized to the sections at 50 C overnight. The slides were then rinsed at room temperature with 2x SSC, rinsed with STE (500 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA) and then incubated at 37 C for 30 min in STE containing 40 µg/ml RNase A. The slides were then incubated 5 min in 2x SSC with 50% formamide at 50 C followed by two 5-min washes at room temperature with 1x SSC and then 0.5x SSC. Immunological detection was performed with sheep anti-Dig Fab fragments conjugated to alkaline phosphatase (Boehringer Mannheim), and color was developed with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium at pH 9.5 in 100 mM Tris-HCl, 50 mM MgCl2, 1 mM levamisole according to the manufacturer’s instructions.


    FOOTNOTES
 
Address requests for reprints to: Daniel W. Carr, Veterans Affairs Medical Center, Mail Stop R&D-8, 3710 Southwest Veterans Hospital Road, Portland, Oregon 97201. E-mail: carrd{at}ohsu.edu

This work was supported in part by NIH Grant HD-36408 (D.W.C.) and United States Department of Agriculture Grant 9702279 (G.E.O.).

1 The nucleotide sequences for murine, bovine, and human AKAP110 have been deposited in the GenBank database under GenBank accession nos. AF093406, AF093407, and AF093408, respectively. Back

Received for publication November 30, 1998. Revision received January 19, 1999. Accepted for publication February 9, 1999.


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