Molecular Characterization of a cDNA That Encodes Six Isoforms of a Novel Murine A Kinase Anchor Protein*

We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (K d ∼ 10 nm) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP.

Protein kinase A (PKA) 1 mediates actions of hormones and neurotransmitters that activate adenylate cyclase (1)(2)(3)(4). Signals carried by cAMP are often directed to effectors that accumulate at discrete intracellular sites (5)(6)(7). Targeting of signals to these sites can be achieved by generating a non-uniform distribution of PKA molecules. This occurs when PKAII␣ and II␤ isoforms are attached to cytoskeleton or organelles by A kinase anchor proteins (AKAPs) (5,6). Prototypic anchor proteins (AKAPs 75, 79, and 150) have a binding site for regulatory (RII) subunits of PKAII isoforms and distinct domains that mediate non-covalent coupling of AKAP⅐PKAII complexes to the microtubule-based dendritic cytoskeleton of neurons and the cortical actin cytoskeleton of non-neuronal cells (5)(6)(7)(8)(9)(10)(11)(12)(13). Both cytoskeletal locations are closely apposed to the plasma membrane. Thus, anchored PKAII is positioned near a signal generator (adenylate cyclase) and multiple PKA substrate/effector proteins (e.g. myosin light chain kinase, microtubuleassociated protein-2, ion channels, serpentine receptors that couple with G s ) (5,6,14). Effector proteins in cAMP signaling pathways can also be associated with organelles, specialized regions of plasma membrane, or sites in cytoskeleton that are separated from adenylate cyclase by substantial distances. Novel RII-binding proteins apparently participate in the assembly of "distal signaling modules" that are associated with mitochondria, Golgi membranes, peroxisomes, and centrioles (5,6,15,16).
Anchored PKAII isoforms may be essential for dissemination of cAMP signals in highly polarized epithelium. Epithelial cells of lung and kidney provide examples of maximally polarized signal transduction, in which hormone-activated adenylate cyclase and downstream target/effectors can be located at opposite ends of the cell. Tight junctions between adjacent cells block communication by diffusible molecules and create two functionally distinct regions of plasma membrane. In epithelial layers of nephrons the basolateral surface is accessible to hormones in plasma and contain receptors and adenylate cyclase; the apical surface is enriched in channel and transporter proteins that mediate absorption/elimination of ions, nutrients, metabolites, and water (17,18). Activities of many channels and transporters that traverse the apical membrane are regulated by hormones that stimulate adenylate cyclase, thereby promoting PKA activation (17)(18)(19)(20)(21). Several considerations suggest that C subunits derived from cytoplasmic PKAs may not mediate trans-epithelial signaling. Signals (cAMP) generated by modest, physiological levels of hormone at the basolateral surface can become weak, diffuse, and insufficient to activate target PKA molecules dispersed (at a relatively low concentration) in cytoplasm. This is due to the transient and intermittent nature of both hormone-release and hormone-mediated stimulation of adenylate cyclase in situ, rapid desensitization of receptor-G protein interactions, dilution of cAMP into the large volume of the cytoplasm, and degradation mediated by cAMP phosphodiesterases. Moreover, studies on knock-out mice and cultured neurons indicate that the ability of PKA isoforms to respond to small changes in cAMP content is often crucial for regulation in vivo (14,22,23). Another constraint is that channels/transporters cluster into patches that constitute only a small portion of the apical membrane surface (24,25). A mechanism that confers increased sensitivity in a specific microenvironment involves concentrating and colocalizing PKAII isoforms with substrate effectors via AKAPs. AKAP⅐PKAII complexes could serve as sensor/transducers that capture highly diffuse and relatively small trans-cellular signals (cAMP generated at the basolateral surface) with enhanced sensitivity (because of the elevation in local PKAII concentration). Signals can then be rapidly amplified and focused directly on the colocalized effector channel (also at high local concentration) to achieve targeted physiological regulation.
The observation that PKAII is associated with fragments of membrane/cytoskeleton derived from the apical portion of kidney epithelium (21) suggests that the preceding model may be operative in polarized tissue. To gain insights into precise physiological roles for anchored PKAII in establishing and mediating polarized signal transduction, it is essential to identify and characterize AKAPs that are (a) expressed in highly polarized epithelial cells, (b) targeted to the vicinity of the apical surface, and (c) able to avidly bind RII (PKAII) in the context of intact cells. We now describe the discovery and characterization of a novel AKAP, which is expressed in lung and kidney (AKAP-KL) and fulfills the listed criteria.

EXPERIMENTAL PROCEDURES
Isolation of cDNAs Encoding AKAP-KL-A full-length cDNA clone encoding murine RII␣ was obtained as described previously for RII␤ (26). The cDNA was subcloned into the yeast bait plasmid pAS1 (CLONTECH) to generate a fusion gene. The gene encodes a hybrid protein in which the yeast GAL4 DNA-binding domain is appended at the N terminus of RII␣. A rat brain cDNA library in the yeast expression plasmid (library vector) pGAD10 was obtained from CLONTECH and screened via the two-hybrid interaction assay as described by Fields (27) and Durfee et al. (28). Growth of auxotrophic yeast on medium lacking Trp, His, and Leu and ␤-galactosidase assays were used to isolate and characterize cDNAs encoding candidate AKAPs. A fragment of cDNA (1.4 kilobase pairs) encoding a portion of a novel RII-binding protein was excised from recombinant pGAD10 (by digestion with EcoRI) and was used as a template to generate a randomprimed, 32 P-labeled probe. This probe was used to screen a 5Ј-stretched mouse brain cDNA library in bacteriophage gt11 (CLONTECH) as indicated in previous papers (29,30). Six positive recombinant phage clones were plaque purified and the cDNAs (0.9 -3.8 kilobase pairs) were subcloned in plasmids pGEM7Z (Promega) and pBluescript (Stratagene) and sequenced.
DNA Sequence Analysis-cDNA inserts were sequenced by a dideoxynucleotide chain termination procedure (31) using T3, T7, and custom synthetic oligonucleotide primers as described previously (30).
Computer Analysis-Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-Intelligenetics software (Intelligenetics, Mountainview, CA) and BLAST programs (32,33) provided by the NCBI server at the National Institutes of Health.
Equilibrium Binding Assay-Aliquots (80 ng) of highly purified partial AKAP-KL protein (residues 354 -741, Fig. 2A) were used for binding assays. Residues 586 -605 constitute the high-affinity RII␣-binding site in AKAP-KL (see "Results"). RII␣ was expressed, purified, and labeled with 32 P as described previously (8,26). Assays were performed in 250 l of buffer A (10 mM Tris-HCl, 50 mM sodium phosphate, pH 8.0, 0.1 M NaCl). 32 P-Labeled RII␣ was varied from 0.1 to 75 nM. Incubations were carried out at 0°C for 90 min. Subsequently, 40 l of a 50% suspension of Ni 2ϩ -chelate Sepharose 4B beads (Talon resin, CLONTECH), which was pre-equilibrated with Buffer A, was added and the incubation continued for 30 min. Next, samples were diluted to 1 ml with buffer A and the beads were pelleted by centrifugation at 2,000 ϫ g for 5 min at 4°C. The beads were washed an 5 additional times by resuspension in 1 ml of buffer A and centrifugation at 2,000 ϫ g at 4°C. Bound 32 P-labeled RII␣ was eluted from the beads in 0.2 ml of 1% SDS and radioactivity was determined in a scintillation counter. 32 P radioactivity in an aliquot of the first supernatant solution was determined to measure the amount of free RII␣.
Growth and Transfection of HEK-293 and AV-12 Cells-Human embryonic kidney fibroblasts (HEK293 cells) and a cell line derived from a hamster subcutaneous tumor (AV-12) were obtained from the American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Full-length AKAP-KL2 and AKAP-KL3 cDNA inserts were excised from recombinant pBluescript plasmids by digestion with SpeI and ClaI and ligated into the pEBG mammalian expression vector (36), which was cleaved with the same enzymes. This placed the cDNAs downstream from a powerful EF 1␣ promoter and a GST gene and upstream from a poly(A) addition signal. Thus, the vector promotes high level expression of GST-AKAP-KL in mammalian cells. Transfections were performed via calcium phosphate precipitation as described previously (13,35). Stable transformants were obtained by selection with 1 mg/ml G418 for 14 days (34).
Expression and Purification of a Fusion Protein That Contains the RII-binding Domain of AKAP-KL-AKAP-KL cDNA (nucleotides 1573 to 2736, Fig. 2A) that encodes amino acids 354 -741 in the anchor protein was cloned into expression plasmid pET14b as described in previous papers (26,37). This enabled abundant synthesis of a Histagged partial AKAP-KL fusion protein in Escherichia coli BL21 (DE3) that was transformed with recombinant plasmid and induced with isopropyl-1-thio-␤-D-galactopyranoside (26,37). Induced bacteria were disrupted in a French press and the soluble His-tagged AKAP-KL protein was purified to near homogeneity by affinity chromatography on Ni 2ϩ -chelate-Sepharose 4B beads (Pharmacia) as described previously (26). Two mg of protein were purified from a 600-ml culture.
Production of IgGs Directed against AKAP-KL-Samples of AKAP-KL fusion protein were injected into rabbits (0.4-mg initial injection; 0.2 mg for each of four booster injections) at Covance Laboratories (Vienna, VA) at 3-week intervals. Serum was collected at 3-week intervals after the first injection.
Deletion and Site-directed Mutagenesis of AKAP-KL-Deletion mutagenesis was performed via polymerase chain reaction as described for AKAP75 and S-AKAP84 (11,15). Amino acid substitutions were introduced into the RII-binding domain of AKAP-KL via site-directed mutagenesis, as described previously (11).
Determination of AKAP-KL Localization by Immunofluorescence Analysis and Immunoperoxidase Histochemistry-HEK293 cells that were stably transfected with an AKAP-KL transgene were fixed and incubated with anti-AKAP-KL antibodies using procedures described by Li et al. (13). AKAP-KL⅐IgG complexes were visualized by incubation with fluorescein isothiocyanate-coupled secondary antibodies and the utilization of a laser scanning confocal microscope system as described previously (13). F-Actin was visualized by its interaction with rhodamine-tagged phalloidin, as previously reported (13). Mice were sacrificed by in situ fixation (12). Sections of kidney and lung were prepared, probed with antibodies directed against AKAP-KL, and stained via an indirect peroxidase procedure as previously reported (12). A black precipitate is formed at sites containing AKAP-KL⅐IgG complexes.

RESULTS AND DISCUSSION
Discovery of a Novel Anchor Protein, AKAP-KL-A cDNA encoding full-length RII␣ was cloned into the yeast plasmid pAS1-CYH2. This recombinant "bait" vector was used to screen a rat brain cDNA library inserted into the yeast "library" vector pGAD10. Interaction of the GAL4 DNA-binding domain-RII␣ fusion protein encoded by the bait vector with RII-binding proteins fused to the activation domain of GAL4 (encoded by library vector) reconstitutes a transcription factor that stimulates GAL4-dependent promoters to transcribe HIS3 and lacZreporter genes. The combination of chimeric genes in plasmids and interaction-dependent HIS3 gene expression enable selection of candidate AKAPs by growth of auxotrophic yeast colonies on Trp Ϫ Leu Ϫ His Ϫ medium. Yeast carrying four different cDNAs were obtained from 10 6 transformants. Each yeast clone produced positive results in ␤-galactosidase assays. Library plasmids were recovered from yeast and amplified in E. coli. Reconstitution assays showed that plasmids encoding candidate AKAPs did not activate HIS3 or lacZ alone, or in combination with irrelevant bait plasmids. In contrast, co-transformation of yeast with selected library plasmids and the original bait plasmid enabled growth on "triple minus" medium and ␤-galactosidase expression. RII-binding assays were performed on samples of total protein from yeast carrying positive, recombinant plasmids. One fusion protein bound ϳ10-fold more 32 Plabeled RII␣ than the others (Fig. 1, lane 8). When the corresponding cDNA was sequenced it yielded a derived protein comprising 415 residues. A translation termination codon was evident, but the 5Ј end of the cDNA corresponded to an open reading frame. Therefore the cDNA insert was used to screen a 5Ј-stretched mouse brain cDNA library in bacteriophage gt11 via standard DNA hybridization procedures. Six overlapping cDNA clones (including one near the full-length clone) were obtained and sequenced ( Fig. 2A). A translation initiation codon was detected and the amino acid sequence of the murine RII-binding protein (named AKAP-KL) was elucidated. The name AKAP-KL was selected because of the expression and highly asymmetric targeting of the anchor protein in epithelial cells of kidney and lung (see below).
A predicted initiator Met codon (nucleotides 514 -516, Fig.  2A) is embedded in the context of a consensus translation start site (ANNATGG) (38). An open reading frame of 884 codons follows the initiator ATG and precedes a translation termination signal at nucleotides 3169 -3171. The upstream cDNA sequence (nucleotides 1-513) includes seven in-frame stop codons, as well as multiple translation termination signals in alternative reading frames. A 3Ј-untranslated sequence of 752 nucleotides was established. Two poly(A) addition signals (AATAAA) are present (nucleotides 3192-3197 and 3367-3372) in this region, but neither seems to be involved in 3Ј end processing. Polyadenylate was not detected at the 3Ј terminus of any of the cDNAs. Moreover, Northern blot analysis indicates that AKAP-KL is encoded by a 9-kilobase mRNA. 2 Thus, the 3Ј-untranslated region of AKAP-KL mRNA may exceed 5 kilobases in length.
Structure-function Relationships in AKAP-KL-AKAP-KL is composed of 885 amino acids and has a calculated M r of 98,000 ( Fig. 2A). The sequence of the acidic polypeptide (pI ϳ 5.0) is not homologous with sequences of previously characterized proteins. However, several domains that could potentially contribute to functional roles for AKAP-KL are evident. The sequence between residues 586 and 605 ( Fig. 2A) of AKAP-KL aligns with the RII-binding site of AKAP75 (11) to yield 45% overall identity (Fig. 3A). A central core of 7 amino acids (Leu 593 to Gln 599 in AKAP-KL) is nearly invariant (86% identity) in the two proteins. Furthermore, five amino acids with large aliphatic side chains, which coordinately regulate RII binding affinity in AKAP75 (11), are conserved in AKAP-KL (Leu 586 , Leu 593 , Val 594 , Ile 598 , and Ile 602 ). A partial AKAP-KL protein (residues 354 -741) that includes the putative tethering site binds both RII␣ (Fig. 3B, lane 1) and RII␤ (not shown). Two approaches verified the functionality of the potential RII-binding site in AKAP-KL. First, a hydrophobic residue (Ile 598 ) predicted to be essential for binding RII (by analogy with AKAP75) was mutated to Ala. This substitution diminished RII binding activity of partial AKAP-KL by Ͼ95% (Fig. 3B, lane 3). A similar effect was observed when the corresponding Ile in AKAP75 was replaced with Ala (11). Mutation of an amino acid predicted to be non-essential (Ala 590 to Ser) had little effect on the tethering of RII (Fig. 3B, lane 2). Partial AKAP-KL proteins that lack residues 586 -605 have no RII binding activity. 2 RII-binding regions in AKAP75 and AKAP-KL are predicted to fold as an amphipathic ␣-helix with one predominantly hydrophobic surface (10). 2 Interactions between this hydrophobic surface and a complementary apolar region near the N terminus of RII subunits stabilize AKAP75⅐RII (PKAII) complexes (26,40). Similar interactions apparently promote the binding of PKAII isoforms by AKAP-KL. Replacement of a critical Ile residue with Ala is a conservative substitution that is unlikely to alter secondary structure or the apolar nature of the RII tethering site. Rather, it appears that a reduction in the size of the hydrophobic binding surface compromises tethering activity (see Ref. 11 for details).
If AKAP-KL mediates accumulation of PKAII in a discrete microenvironment within cells, then anchor protein⅐RII complexes should have a low dissociation constant, indicative of stable protein-protein interactions. Purified His-tagged AKAP-KL fusion protein (residues 354 -741) was incubated to equilibrium with various concentrations of 32 P-labeled RII␣. Subsequently, AKAP-KL⅐RII␣ complexes were recovered on Ni 2ϩ -chelate Sepharose 4B beads and amounts of bound and free radiolabeled ligand were measured in a scintillation counter. Analysis of typical results (Fig. 3C) yielded a K d value of 9.5 nM, indicating that AKAP-KL avidly binds RII (PKAII). In many tissues the total RII concentration is ϳ150 nM (41). If the concentration of AKAP-KL is assumed to be 10 -100 nM, then the K d and mass law considerations (42) indicate that Ն85% of the tethering sites will be occupied by RII (PKA) isoforms.
Two segments of AKAP-KL (residues 246 -316 and 729 -766, Fig. 2A) are predicted to be coiled-coil domains (43). Such structures mediate homomeric and heteromeric protein-protein interactions. A segment of AKAP-KL (residues 274 -312) is composed of Gln (79%) interspersed with occasional Leu residues. The sequence LQQQQ appears 5 times within this region. Studies on proteins and model peptides indicate that Gln-rich domains serve as "zippers" that interlock polypeptides into oligomeric structures (44). Although the LQ 4 motif has not been previously described as a specific entity, it is established that Leu side chains are crucial components of zippers (actually coiled-coil regions) that facilitate protein-protein interactions (45). Thus, it is possible that the LQ 4 1. Expression of RII␣-binding proteins in yeast. Samples of total protein (35 g) from colonies of transformed yeast were subjected to electrophoresis in a denaturing polyacrylamide gel (10%). Subsequently, size-fractionated proteins were transferred to an Immobilon P (Millipore) membrane and probed with 32 P-labeled RII␣ (overlay assay, see "Experimental Procedures") to determine the binding activities of the fusion proteins. Lanes 1, 2, and 8 received fusion proteins from yeast transformed with recombinant pGAD10 plasmids that encode three distinct RII-binding proteins, which were identified in the 2-hybrid interaction screen. Lanes 4 and 5 contained proteins from non-transformed yeast and yeast containing the RII␣ cDNA in the bait vector, respectively. Lanes 6 and 7 received proteins from yeast transformed with pGAD10 plasmids encoding proteins that do not interact with RII␣. The sample in lane 8 corresponds to a fragment AKAP-KL. An autoradiogram is shown. Only the relevant part of the blot is presented.
functions of coiled-coil regions in AKAP-KL can be rigorously evaluated in future studies by employing a combination of mutagenesis, transfection/expression, biochemical analysis, and immunolocalization techniques.
Six Isoforms of AKAP-KL Are Produced in Mammalian Cells-Sequencing of 3Ј ends of AKAP-KL cDNAs revealed two alternative modes of AKAP-KL mRNA splicing. In the first instance, a splice donor sequence at nucleotide 2878 ( Fig. 2A) is joined to an acceptor site at position 3097, thereby deleting 218 nucleotides in cDNA. This deletion shifts the reading frame after codon 789 so that the succeeding 21 nucleotides encode a novel, C-terminal hexapeptide (Fig. 2B) and a translation termination signal. As a result, the C terminus of the anchor protein is truncated by 90 residues. AKAP-KL proteins that lack 90 C-terminal residues are designated AKAP-KL3; AKAP-KL proteins that retain this region are designated AKAP-KL1. A second mode of RNA splicing joins a donor sequence at nucleotide 3058 ( Fig. 2A) with the acceptor site at nucleotide 3097. This splicing reaction excises 13 codons (for amino acids 849 -861) and does not alter the reading frame (Fig. 2B). Anchor proteins that lack residues 849 -861 are named AKAP-KL2.
Further diversity in AKAP-KL structure is introduced by utilization of either of two translation start codons. Like codon 1 in Fig. 2A, Met codon 125 is included within the context of a consensus translation initiation sequence (ANNATGG, nucleotides 883-889). Both start sites are used in cultured cells and in vivo (see Fig. 4, below). Thus, AKAP-KL proteins can differ by retention (form A) or deletion (form B) of a 124-residue N-terminal domain. Together, alternative splicing of AKAP-KL mRNA and utilization of two translation start codons generate 6 discrete AKAP-KL isoforms. Sizes and nomenclature for AKAP-KL isoforms are presented in Table I.
The physiological significance of various AKAP-KL isoforms remains to be determined. However, the ability of individual cells to produce up to 6 related, but distinct AKAPs from a single gene suggests mechanisms for the diversification and specialized adaptation of PKAII-mediated signal transduction. For example, different AKAP-KL isoforms may be targeted to distinct intracellular locations. (All forms of the anchor protein contain the RII tethering site.) Differences in stability (t1 ⁄2 ) among anchor protein variants could alter the concentration of immobilized (versus cytoplasmic) PKAII, thereby shifting the focus of PKAII-mediated signaling from one compartment to  Table I). Residues 1-780 are identical in each of the anchor proteins. The unique Cterminal hexapeptide in AKAP-KL3 isoforms is underlined.

FIG. 2 continued
another. Various AKAP-KL isoforms might subserve scaffolding functions and contain different combinations of co-clustered PKA substrates or modulators of PKA activity. Splicing of AKAP-KL mRNA and selection of translation initiation codons may also be subject to cell-specific and/or developmental regulation. Differential expression of anchor protein isoforms provides a potential mechanism for changing the localization and functions of PKAII isoforms in response to environmental stimuli and developmental cues. Addition or subtraction of proteinbinding sites by splice site selection could produce AKAP-KL isoforms that integrate PKAII with different assemblies of signal transducing proteins.
Generation of AKAP diversity by regulated, differential mRNA splicing may be a frequently used mechanism. S-AKAP84 and AKAP121 (D-AKAP-1) mRNAs are derived from a single gene transcript by alternative splicing (37,46). The smaller anchor protein (S-AKAP84) is uniquely produced in male germ cells (15,37), wherein it recruits PKAII␣ to specialized mitochondria. mRNA for the larger isoform (AKAP121) is generated by a splicing reaction that occurs in many somatic cells. In germ cells this splicing reaction is partly suppressed (37). Inhibition of splicing results in retention of an in-frame translation termination codon and the production of a 58-kDa RII-binding protein (S-AKAP84). AKAP121 (M r ϭ 92,000) and S-AKAP84 share identical RII tethering sites and N-terminal targeting domains that direct the anchor proteins to the outer membrane of mitochondria. However, the functionality of the larger AKAP is diversified by a C-terminal extension that contains at least two motifs involved in the binding of RNA molecules (37).
Preparation and Specificity of Antibodies Directed against AKAP-KL-AKAP-KL cDNAs were cloned from a brain cDNA library. Since a high proportion of the complete constellation of mammalian genes is expressed in the central nervous system, the efficiency of screening is maximized. However, isolation of reverse transcripts of AKAP-KL mRNAs from this library does not imply that brain is the principal site of accumulation of the anchor protein. To determine the tissue and cellular distributions, as well as functions of AKAP-KL it is essential to generate highly specific anti-AKAP-KL IgGs.  imidazole (1 g, lane 3) were size-fractionated by denaturing electrophoresis. A 10% polyacrylamide gel stained with Coomassie Blue is shown. The partial AKAP-KL protein has an apparent M r of 50,000. B, cytosol (100,000 ϫ g supernatant solution) and two particulate fractions (P1, 10,000 ϫ g pellet; P2, 100,000 ϫ g pellet) were prepared from mouse kidney as described previously (8,12). Samples (35 g) of cytosolic, P1 and P2 proteins were size-fractionated in a denaturing polyacrylamide gel (9%) and transferred to an Immobilon P membrane. Lanes 1 and 5 received cytosolic proteins; lanes 2 and 6 contained P2 proteins; lanes 3, 4, and 7-9 were loaded with proteins from the P1 pellet. Lanes 1-4 from the Western blot were incubated with preimmune serum (1:1000 dilution) and lanes 5-8 were probed with antiserum directed against AKAP-KL (1:2000 dilution) as indicated under "Experimental Procedures." Excess purified antigen (3 g) was present when lanes 4 and 8 were probed with preimmune serum and antiserum. The immunoblots were developed by an enhanced chemiluminescence procedure and signals were recorded on x-ray film (see "Experimental Procedures"). RII-binding proteins in the P1 pellet were identified and characterized by the overlay binding assay (lane 9). An autoradiogram is shown. A fragment (1164 base pairs) of AKAP-KL cDNA encoding amino acids 354 -741 ( Fig. 2A) was amplified by the polymerase chain reaction using 5Ј and 3Ј primers that introduced NdeI and BamHI restriction sites, respectively. This enabled cloning of the cDNA fragment into the pET14b expression plasmid. The inserted cDNA lies downstream from a bacteriophage T7 promoter and DNA encoding a fusion peptide composed of 20 amino acids. Included in the fusion peptide are six consecutive His residues, which constitute a Ni 2ϩ -binding site. E. coli BL21 (DE3) was transformed with recombinant pET14b plasmid and induced to synthesize fusion protein with 1 mM isopropyl-1thio-␤-D-galactopyranoside. After lysis of bacteria in a French press, the soluble, partial AKAP-KL fusion protein was purified to near homogeneity by affinity chromatography on a metal chelate-Sepharose 4B resin (Fig. 4A).
Antibodies directed against the partial AKAP-KL protein were produced in rabbits. The antibodies will bind with all AKAP-KL isoforms (Table I) because the cDNA fragment encoding the antigen begins downstream from the second translation initiation codon and terminates upstream from donor sites for alternative mRNA splicing. Western immunoblot analysis revealed that two major AKAP-KL polypeptides (with apparent M r values of 105,000 and 120,000) accumulate in the 10,000 ϫ g pellet fraction (P1) of mouse kidney homogenates (Fig. 4B, lane 7). Two minor AKAP-KL proteins (apparent M r ϭ 115,000 and 130,000) are also detected. Lower levels of the same proteins are evident in the 100,000 ϫ g particulate fraction (P2), whereas cytosol lacks the RII-binding protein (Fig.  4B, lanes 5 and 6). Thus, AKAP-KL is tightly associated with cytoskeleton and/or organelles. Polypeptides with the same M r values avidly bind 32 P-labeled RII␣ (Fig. 4B, lane 9). AKAP-KL isoforms (Table I), like other AKAPs (11,15), are acidic proteins that exhibit aberrantly large M r values in denaturing gels.
Expression of AKAP-KL Is Tissue-specific and Isoform-selective-Analysis of P1 fractions from various tissues disclosed that AKAP-KL is abundantly expressed in lung (Fig. 5A, lane  1). In contrast, the anchor protein is either absent or produced at very low levels in multiple tissues, including liver, heart, and cerebral cortex (Fig. 5A, lanes 3-5). Moderate levels of AKAP-KL were detected in thymus and cerebellum. AKAP-KL isoforms observed in cerebellum (Fig. 5A, lane 6) do not comigrate with anchor proteins that accumulate in lung. Thus, expression of the AKAP-KL gene is tightly regulated. Accumulation of AKAP-KL polypeptides is restricted to certain cell/ tissue types and anchor protein isoforms that predominate in a given tissue are evidently determined by a combination of regulated AKAP-KL mRNA splicing and utilization of two translation initiation codons.
It is possible to identify AKAP-KL isoforms expressed in vivo by comparison with AKAPs encoded by transgenes in transfected cells. Hamster AV-12 cells lack endogenous AKAP-KL. AV-12 cells transfected with an AKAP-KL2 transgene accumulate anchor proteins with apparent M r values of 115,000 and 130,000 (Fig. 5B, lane 3). In contrast, an AKAP-KL3 transgene programs the synthesis of 105-and 120-kDa RII-binding proteins (Fig. 5B, lane 2). Deletion of 120 codons at the 5Ј end of AKAP-KL3 cDNA forces the exclusive utilization of the second initiator Met codon and results in the production of a single anchor protein with an apparent M r of 105,000 (Fig. 5B, lane 1). Comparison of anchor proteins synthesized in lung tissue (Fig.  5B, lane 4) with those expressed in transfected cells disclosed that AKAP-KL3A and AKAP-KL3B (Table I) (Table I) are synthesized in lung. The principal anchor proteins in cerebellum (see Fig. 5A) were identified as AKAP-KL2A and AKAP-K2B by applying the same methodology.
Properties of AKAP-KL in Vitro and in Cells-Extraction with buffers containing 0.5% Triton X-100 failed to solubilize the anchor protein, indicating that is not embedded in a lipid bilayer (Fig. 6A). Buffers containing 0.5% sodium deoxycholate (which disrupts association of proteins in cytoskeleton) efficiently solubilize AKAP-KL. Confocal immunofluorescence microscopy of HEK293 cells that were stably transfected with cDNA encoding full-length AKAPs KL2A and 2B revealed the intracellular distribution of the anchor protein. AKAP-KL accumulates in regions of cortical cytoskeleton that appear as projections or large clusters of antigen (Fig. 6B). These structures are not present in control HEK293 cells or cells transfected with unrelated transgenes (13). In contrast, AKAP75 is dispersed throughout the cortical cytoskeleton in the same cells (13). Thus, AKAP-KL appears to be targeted to specific microenvironments in cytoskeleton. It is possible that AKAP-KL actively promotes assembly/organization of specialized structures via interactions with other proteins in cytoskeleton. The differential clustering of AKAP-KL in subdomains of cytoskeleton in HEK293 cells may also reflect properties involved in targeting of the anchor protein to the vicinity of the apical surface of polarized epithelial cells (see below).
Cytochalasin D disrupts the F-actin network, causes marked cell rounding and elicits a redistribution of AKAP-KL to two sites (Fig.  6C). In part, the anchor protein is dispersed along the cell periphery; the remainder of AKAP-KL is aggregated in large internal structures. Double immunostaining disclosed that both peripheral and internal AKAP-KL are associated with F-actin in cytochalasintreated cells (Fig. 6D). F-actin is distributed evenly in the cortex of  Table I for nomenclature and sizes of the anchor protein isoforms.) Lane 4 received P1 proteins isolated from a lung homogenate. untreated, transfected cells (e.g. see Ref. 13). Thus, AKAP-KL binds with actin or actin-associated proteins in cytoskeleton. However, cytochalasin D-sensitive interactions with other (cytoskeletal?) proteins apparently restrict and enrich AKAP-KL in discrete microenvironments. A speculation is that such mechanisms might contribute to assembly of polarized distal signaling modules that include anchored PKAII.
AKAP-KL Is Targeted to the Apical Surface of Polarized Epithelial Cells-Anti-AKAP-KL IgGs were used in an indirect immunoperoxidase staining procedure to determine the location of AKAP-KL in sections of rat kidney. The anchor protein is expressed in epithelial cells and accumulates exclusively at the apical surface, which abuts the lumen (e.g. see epithelial cells in proximal tubules, Figs. 7, A and B). RII␣ is also concentrated at the apical surface of epithelial cells in renal proximal tubules (21). In lung, AKAP-KL accumulates in long, linear structures just below the lumenal surface of alveolar epithelial cells (Fig. 7C). In parallel, a substantial amount of RII is also concentrated in the elongated structures near the apical surface of alveolar epithelial cells (Fig. 7D). The preceding results suggest that AKAP-KL and RII are co-localized and co-enriched in two types of polarized epithelium. Confirmation of this working hypothesis will ultimately require high-resolution, immunoelectron microscopy and manipulation of the system by molecular genetics (e.g. disrupting AKAP-KL⅐RII complexes by expressing AKAP-KL mutants that bind RII, but are mis-targeted, or by ablating AKAP-KL gene expression) in future studies. However, the present observations support the idea that AKAP-KL incorporates PKAII into distal signaling modules at sites near junctions of cytoskeleton and apical plasma membrane. Such modules might include PKA substrate/effector proteins (e.g. transporters) which span the apical membrane. This mode of organization can greatly facilitate cAMP-mediated trans-epithelial signal transduction.
AKAP-KL Binds RII with High Affinity in Intact Cells-The pEBG expression vector (36) was used to assess the ability of the tethering domain AKAP-KL to sequester RII in intact cells. pEBG contains a powerful EF 1␣ promoter that lies upstream from the glutathione S-transferase (GST) gene and a 3Ј multiple cloning region. AKAP-KL cDNA was inserted, in-frame with the GST gene, and the vector was co-transfected with R C /CMV (an expression plasmid which contains the neo R gene under control of an SV40 promoter) into hamster AV-12 cells. Stable cell lines expressing GST-AKAP-KL were isolated by selection with G418. Typically, ϳ80% of GST-AKAP-KL appeared to be anchored, whereas 20% was present in cytosol. Cytosolic GST⅐AKAP-KL complexes were purified to near homogeneity by affinity chromatography on GSH-Sepharose 4B. Western blot analysis revealed that a large proportion of avail-able cytosolic RII was complexed with the anchor protein (Fig.  8). Two conditions were varied to exclude the possibility that AKAP-KL⅐RII complexes assembled after cell homogenization. Since AKAP-KL⅐RII complexes dissociate very slowly at 4°C, 2 cells were disrupted: (a) in a volume of lysis buffer that was increased 20-fold to reduce concentrations of free RII (PKAII) and anchor protein or (b) in a standard volume of buffer that contained a vast excess (15 g/ml) of His-tagged AKAP-KL partial protein (see Fig. 4A). The partial anchor protein avidly binds RII (Fig. 3C), but is not sequestered by GSH-Sepharose 4B resin. Neither dilution nor post-lysis competition with an RII-binding protein altered the results obtained in Fig. 8 (data  not shown). Thus, stable AKAP-KL⅐RII complexes are efficiently produced in the environment of the internal milieu of intact cells.
Conclusions and Implications-AKAP-KL isoforms are differentially expressed and selectively targeted to apical regions of polarized lung and kidney epithelial cells. This family of novel anchor proteins efficiently sequesters RII (PKAII) in vitro and in situ. AKAP-KL proteins may be involved in (a) establishing polarity in trans-epithelial signaling systems and (b) creating/organizing distal PKA⅐effector complexes that receive, amplify, and focus trans-cellular signals carried by cAMP. Generation of six AKAP-KL isoforms by post-transcriptional processes creates substantial anchor protein diversity and suggests possible mechanisms by which intracellular localization of PKAII isoforms may be modulated in different cell types and in response to environmental stimuli.