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
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ABSTRACT
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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.
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INTRODUCTION
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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
and RIß
subunits are predominantly localized in the acrosomal segment of the
head whereas RII
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.
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RESULTS
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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. 1
)) 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
3.010.0. 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.
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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
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 289294
(AGGATGG) meets all the requirements for a consensus start
site (18, 19). The 5'-upstream sequence, nucleotides 1288, contains
10 inframe stop sites, precluding the possible use of another ATG
upstream. A termination site is found at nucleotides 28842886 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. 2
. 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. 2
), 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.
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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. 3
). 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. 3C
). 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 (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.
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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. 4
, A and B). Deletion of the
C-terminal half of the protein (amino acids 471864) had no effect on
RII binding, while removal of the N-terminal half (amino acids
1470) 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 (1864) or truncated fragments 1470,
471864, 1350, 1164, and 1117 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 (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
(127140) peptide (FYANRLTNLVIAMA).
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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 127140, 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. 4C
).
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. 5A
).
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
-helix, all of the conserved
positions align to one face on the
-helix (Fig. 5B
). 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
-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. 5C
).

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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.
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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. 6
), 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. 6
) 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. 5A
) 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.
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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. 7
), 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.
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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. 8A
). 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. 8B
). Late
spermatids also exhibited no detectable expression of AKAP110 (Fig. 8B
). AKAP110 sense riboprobes gave no detectable signals (Fig. 8C
).

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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.
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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. 9
, A and A').
Spermatozoa immunostained with anti-AKAP110 exhibited specific
fluorescence along the entire flagellum (Fig. 9
, A and A'). No
fluorescence was detectable in spermatozoa stained with identical
dilutions of preimmune serum (Fig. 9
, 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.
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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. 3
and 4
); and 3)
Antibodies against a synthetic polypeptide within AKAP110 recognize
both the recombinant and native AKAP110 (Fig. 3
).
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. 7
). In
situ hybridization analysis detected AKAP110 only in round
spermatids (Fig. 8
), 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. 1
). 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. 124141) 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. 6
), 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. 1180) 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. 7
) 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. 3
), 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. 1
and 4C
, this manuscript, and Fig. 1C
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. 5
) 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
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
|
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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
-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 310
(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. 1A
, 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
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
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
(
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 448459 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
-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
(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
manufacturers 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. 
Received for publication November 30, 1998.
Revision received January 19, 1999.
Accepted for publication February 9, 1999.
 |
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