Journal of Cell Science 113, 2715-2724 (2000) 2715 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1538

Assembly of multiple -containing complexes in the kidney

Nellie Y. Loh1, Sarah E. Newey1, Kay E. Davies1,2,* and Derek J. Blake1 1Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK 2MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK *Author for correspondence (e-mail: [email protected])

Accepted 22 May; published on WWW 10 July 2000

SUMMARY

Dystrophin is the key component in the assembly and whilst α-dystrobrevin-1 containing complexes are found in maintenance of the -associated complex endothelial and cells. Furthermore, these (DPC) in . In kidney, , an complexes are maintained even in the absence of all integral component of the DPC, is involved in kidney dystrophin isoforms. Thus our data suggest that the epithelial morphogenesis, suggesting that the DPC is functions and assembly of the dystrophin-like complexes in important in linking the extracellular matrix to the internal kidney differ from those in skeletal muscle and implicate a cytoskeleton of kidney epithelia. Here, we have investigated protein other than dystrophin as the primary molecule in the molecular architecture of dystrophin-like protein the assembly and maintenance of kidney complexes. Our complexes in kidneys from normal and dystrophin- findings also provide a possible explanation for the lack of deficient mice. Using isoform-specific antibodies, we show kidney pathology in Duchenne muscular dystrophy that the different cell types that make up the kidney patients and mice lacking all dystrophin isoforms. maintain different dystrophin-like complexes. These complexes can be broadly grouped according to their dystrobrevin content: β-dystrobrevin containing complexes Key words: Dystrobrevin, Dystrophin, Utrophin, Syntrophin, Dp71, are present at the basal region of renal epithelial cells, Kidney, Epithelium

INTRODUCTION kinases (MAST205 and SAST; Lumeng et al., 1999). The importance of this signalling role in muscle function is Dystrophin, the product of the Duchenne muscular dystrophy confirmed by the finding that α-dystrobrevin-deficient mutant (DMD) gene, is part of a multi-protein complex that is required mice display muscular dystrophy and greatly reduced levels of for normal muscle function. Although many components of nNOS from the , despite the fact that distribution this complex are restricted to muscle, the cytoplasmic of all other components of the DPC remain unchanged (Grady components of the dystrophin-associated protein complex et al., 1999). (DPC) are widely expressed. The cytoplasmic complex There is an increasing body of evidence showing that the comprises members of the dystrophin, dystrobrevin and DPC plays a role in non-, in particular kidney. syntrophin protein families. In skeletal muscle, the DPC is Firstly, α-dystroglycan, the extracellular component of the assembled around dystrophin. This scaffold links the DPC and a binding partner of several components of the cytoskeleton to the basement membrane via the transmembrane extracellular matrix, is present in tubular and glomerular protein β-dystroglycan and anchors the syntrophins and α- basement membranes of the kidney (Durbeej et al., 1995; dystrobrevin to the muscle membrane. Loss of dystrophin leads 1998). Antibody perturbation experiments in vitro have shown to disassembly of the complex and muscle degeneration that this protein is important in kidney epithelial (reviewed in Blake and Davies, 1997). morphogenesis (Durbeej et al., 1995). Secondly, several In addition to its structural role, the cytoplasmic components isoforms of dystrophin and utrophin are expressed in kidney of the DPC are involved in intracellular signalling. There are (Durbeej et al., 1997; Deconinck et al., 1997; Grady et al., three muscle-specific isoforms of α-dystrobrevin (1-3), of 1997; Lidov et al., 1998). Finally, β-dystrobrevin, a member of which α-dystrobrevin-1, the largest isoform, is a substrate for the dystrobrevin protein family that is restricted to non-muscle tyrosine kinases (Wagner et al., 1993; Blake et al., 1996; tissues, is most abundantly expressed in brain and kidney, and Balasubramanian et al., 1998; Nawrotzki et al., 1998). The forms a complex with dystrophin isoforms and syntrophin in syntrophins (α, β1 and β2) themselves bind to signalling liver and brain (Peters et al., 1997b; Blake et al., 1998, 1999; such as neuronal (nNOS; Puca et al., 1998). This protein is also part of a utrophin protein Brenman et al., 1996), voltage-gated sodium channels (Gee et complex at the basolateral membrane of polarised epithelial al., 1998), stress-activated protein kinase 3 (Hasegawa et al., cells (Kachinsky et al., 1999). 1999), and a family of microtubule-associated serine/threonine In this paper, we have investigated the distribution and 2716 N. Y. Loh and others organisation of dystrophin-like complexes in the kidneys of Triton fraction, in the following manner: 2-5 µg of antibodies were normal and dystrophin-deficient mice. Our findings reveal that, added to 1 mg protein in a final volume of 1 ml. Following an unlike skeletal muscle where only two dystrophin complexes overnight incubation at 4°C on a blood mill, 100 µl of Magna Bind are present, in kidney there are at least six different dystrophin- goat anti-rabbit IgG (Pierce and Warriner, equilibrated in HB buffer like complexes that are distributed in a cell-type specific + 1% Triton) was added and the mixture was incubated overnight as manner. Interestingly, unlike the muscle DPC, which is before. The beads were then washed extensively in HB + 0.2% Triton. Bound proteins were eluted by boiling for 10 minutes in treatment disrupted in the absence of dystrophin, the localisation and buffer (75 mM Tris-HCl, pH6.8, 4 M urea, 3.8% (w/v) SDS, 20% interactions of kidney dystrobrevins and syntrophins are (v/v) glycerol, 5% (v/v) 2-mercaptoethanol). Immunoprecipitated maintained in the absence of all known dystrophin isoforms proteins were separated on SDS-polyacrylamide gels, transferred onto and this is not due to the upregulation of utrophin, a nitrocellulose membranes, and detected with antibodies using dystrophin-related protein. Taken together our data indicate standard methods. differences in the roles and assembly of the dystrophin-like complexes in kidney and the muscle DPC, and implicate Immunohistochemistry another protein, perhaps dystrobrevin, as the key regulator in Kidneys from adult (>2 months old) C57BL/10 and mdx3Cv mice were the assembly and maintenance of the kidney complexes. embedded in embedding compound (Cryo-M-Bed, Bright) and frozen in liquid nitrogen-cooled isopentane. 8-µm serial sections were cut on a cryostat (Model OTF, Bright) cooled to −20°C, collected onto − MATERIALS AND METHODS SuperFrost Plus (BDH) slides, air-dried and stored at 70°C. Prior to immunofluorescence studies, frozen slides were air-dried at room temperature. Tissue sections were then blocked in 10% (v/v) foetal Polyclonal antibodies calf serum in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris- The antibodies utilized in this paper and their specificity are listed in HCl, pH 7.5) for 20 minutes before incubating in primary antibodies Table 1. diluted in TBS for 1 hour at room temperature. The slides were then The β417 antibody was raised by immunising New Zealand White subjected to two 5 minute washes in TBS and incubated in lissamine rabbits with keyhole-limpet-hemocyanin-coupled synthetic peptide rhodamine-conjugated donkey anti-rabbit secondary antibodies NH2-CAEAGNMTRPPTDASFN, which corresponds with amino (Jackson ImmunoResearch) at 1:50 dilution for 1 hour at room acids 414-429 of β-dystrobrevin (Genosys). The generation of anti- temperature. Slides were finally washed twice in TBS for 5 minutes, syntrophin antibodies was as described by Peters et al. (1997a). 2045 mounted with one drop of Vectashield (Vector Laboratories), and (anti-β2-syntrophin), 2688 (anti-α1-syntrophin) and 2689 (anti-β1- viewed under a fluorescence microscope (Leica). syntrophin) were raised against the keyhole-limpet-hemocyanin- Antibodies were tested for specificity of labelling by preincubating coupled synthetic peptides NH2-CSGSEDSGSPKHQNTTKDR, each antibody with 100 µM of its antigenic peptide 30 minutes before NH2-CRQPSSPGPQPRNLSEA and NH2-CRLGGGSAEPLSSQSF- labelling. SFHRDR, respectively. Each antibody was affinity purified against the immunising peptide immobilised on a SulfoLink column (Pierce) β according to the manufacturer’s protocol. The -dystrobrevin-specific RESULTS antibody β521 was described in Blake et al. (1999). The βCT-FP antibody, which recognises β-dystrobrevin and α-dystrobrevin-1 and -2, and the α1CT-FP antibody, which specifically recognises α- Expression of the dystrophin and dystrobrevin dystrobrevin-1, were described previously (Blake et al., 1998). The protein families in the kidney anti-dystrophin antibody P6 was raised against the distal rod domain To determine the presence of members of the dystrophin of dystrophin and detects all dystrophin isoforms except Dp71 protein family in kidney, western blots were performed with a (Sherratt et al., 1992). The anti-dystrophin antibody 2166 was raised panel of antibodies (Table 1). β-Dystrobrevin, α-dystrobrevin- against the last 17 amino acids of mouse dystrophin and detects all 1, utrophin, dystrophin and Dp71 were all expressed in kidney. dystrophin isoforms. The anti-dystrophin antibody 2401 was raised Of the dystrophin isoforms, the two C-terminal splice forms of against the alternative C terminus of dystrophin generated in the absence of exon 78 (∆C). The anti-utrophin antibody URD40 was raised against the distal rod domain of mouse utrophin and specifically detects utrophin. Purification and characterization of the 2166, 2401 Table 1. Polyclonal antibodies and their specificity and URD40 antibodies were as described in Blake et al. (1999). Antibody Specificity β β α α Immunoprecipitation CT-FP -dystrobrevin, -dystrobrevin-1 and -dystrobrevin-2 β417 β-dystrobrevin only Proteins were prepared according to the method of Kramarcy et al. β521 β-dystrobrevin only (1994) with minor modifications. Frozen kidneys and liver from α1CT-FP α-dystrobrevin-1 only C57BL/10 and mdx3Cv mice were homogenised in 10 ml ice-cold P6 Dystrophin, Dp260, Dp140, Dp116 homogenisation buffer (HB) containing 0.4 M NaCl, 10 mM sodium 2166 All dystrophin isoforms with the C terminus encoded by phosphate, pH 7.8, 5 mM EDTA and protease inhibitors (Complete, exon 78 Roche). After a 10-minute centrifugation at 4°C, 12,000 g, the 2401 All dystrophin isoforms with the alternative C terminus ∆ supernatant was carefully removed and the pellet was rehomogenised ( C)* URD40 Utrophin only in 10 ml ice-cold HB buffer with Triton X-100 added to a final 2688 α-syntrophin only concentration of 1% (v/v). The homogenates were incubated on ice 2689 β1-syntrophin only for 15 minutes and centrifuged as before. The supernatant from the 2045 β2-syntrophin only second centrifugation was carefully removed and the pellet was resuspended in 10 ml HB buffer by homogenisation. 5 µl of each List of primary antibodies used in our western blot, immunoprecipitation fraction was analysed by immunoblotting to determine which fraction and immunofluorescence studies, and the proteins that are recognised by each contained the proteins of interest. antibody. Immunoprecipitations were carried out using the HB buffer +1% *D. J. Blake and M. A. Benson, manuscript in preparation. Dystrobrevin complexes in kidney 2717

and peritubular capillaries in the renal cortex, but not in cortical renal tubules (Fig. 3A,C). In addition, α-dystrobrevin-1 was found in vascular and smooth muscle cells (Fig. 3B), while utrophin was seen in collecting ducts (Fig. 3D). Of the anti-dystrophin antibodies used, 2166-staining was restricted to smooth muscle cells of large blood vessels and the renal papilla (Fig. 3E,F). By contrast, 2401, which was raised against the alternative C terminus of dystrophin (∆C), labelled the basal membranes of cortical renal tubules, Bowman’s

Fig. 1. Expression of the dystrophin protein family in normal adult mouse kidney. Proteins from whole mouse kidneys were fractionated on an 8% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The blot was probed with β521 (lane 1), βCT-FP (lane 2), α1CT-FP (lane 3), URD40 (lane 4), 2166 (lane 5), 2401 (lane 6) and P6 (lane 7). The positions of the marker proteins (in kDa) are shown.

Dp71 were the most abundantly expressed (Dp71 and Dp71∆C, Fig. 1). By contrast, no Dp140 was detected, probably because this dystrophin isoform is only expressed during kidney development (Durbeej et al., 1997; N. Y. Loh et al., manuscript in preparation). To establish the location of these proteins in the kidney, the same panel of antibodies was used for immunofluorescence examination on kidney cryosections. The localisation of β- dystrobrevin in kidney was determined using two β- dystrobrevin-specific antibodies, β417 and β521 (Blake et al., 1998, 1999). On kidney sections, these antibodies labelled the basal membrane of epithelial cells in renal tubules (Fig. 2A,D), collecting ducts (Fig. 2G), Bowman’s capsules (Fig. 2A,D) and glomeruli (Fig. 2A,D). Vascular and smooth muscle cells were not labelled (Fig. 2H). The luminal staining observed in large blood vessels is due to autofluorescence of elastin and is seen in large blood vessels stained with secondary antibody alone (Fig. 2J). The specificity of labelling for both antibodies was confirmed by competition with the immunising peptides (Fig. 2B,F). The non-membrane staining observed is due to non- specific staining since a similar result is observed with the secondary antibody alone (Fig. 2I). Competition with the non- immunising peptide had no effect on antibody-labelling (Fig. 2C,E). Since both β417 and β521 gave very similar staining patterns in kidney, all remaining experiments were performed using β521. α-Dystrobrevin-1 and utrophin were detected in glomeruli

Fig. 2. Immunolocalisation of β-dystrobrevin in adult mouse kidney. Mouse kidney cryosections were stained with the following antibodies against β-dystrobrevin: β417 (A) and β521 (D,G,H). The specificity of staining of each antibody was confirmed when preincubation with the immunising peptide abolished all staining (B, Ab β417+β417pep; F, Ab β521+β521pep) while incubation with an unrelated peptide (C, Ab β417+β521pep; E, Ab β521+β417pep) did not. (I,J) Kidney cryosections incubated with the secondary antibody alone. β-Dystrobrevin was detected in the renal tubules, glomeruli, Bowman’s capsules (A,D) and collecting ducts (G), but not in the large blood vessels (H). Arrowheads, glomerulus; arrows, cortical renal tubules. Bar, 100 µm. 2718 N. Y. Loh and others capsules, and to a lesser extent, the mesangium (Fig. 3G,H), in antibody, which detects only dystrophin in kidney, labelled the addition to (Fig. 3H, inset). The P6 major blood vessels, arterioles and the papillal smooth muscle cells (data not shown). This suggests that dystrophin is only present in these regions in the kidney whilst the isoform present in the cortical renal tubules, Bowman’s capsule and mesangium is Dp71∆C. 2166-staining is attributable to the immunolocalisation of dystrophin, Dp71, or both. Thus, β-dystrobrevin and α-dystrobrevin-1 are differentially distributed in kidney. β-dystrobrevin colocalises with Dp71∆C in cortical renal tubules and Bowman’s capsules, and with utrophin in collecting ducts. By contrast, α-dystrobrevin-1 colocalises with utrophin in peritubular capillaries and glomeruli, and with dystrophin and Dp71 in blood vessels and smooth muscle. These data suggest that several different dystrophin-like complexes may be present in the kidney. Molecular organisation of dystrobrevin containing complexes in the kidney To determine the molecular organisation of dystrobrevin containing complexes that are present in the kidney, detergent- solubilized protein extracts from kidneys were immunoprecipitated with a panel of antibodies against members of the dystrophin protein family and analysed by western blotting. A parallel experiment was performed using detergent-solubilized liver extracts as a control since β- dystrobrevin containing complexes have been successfully purified from this tissue (Peters et al., 1997b; Blake et al., 1999). β-Dystrobrevin copurified with utrophin, Dp71 and Dp71∆C, but not with α-dystrobrevin-1 in kidney (Fig. 4A), and reciprocal immunoprecipitation experiments confirmed these associations (Fig. 4C-E). Dp71, however, was not detected in kidney β521 immunoprecipitate (Fig. 4D,kidney, lane 2), probably because of its relatively low abundance in this tissue compared with other β-dystrobrevin interacting proteins. By contrast, α-dystrobrevin-1 only coimmunoprecipitated with utrophin (Fig. 4B), showing that α-dystrobrevin-1 forms a complex with utrophin and not Dp71 or Dp71∆C. The dystrobrevins formed similar associations in liver, the differences being the low abundance of α-dystrobrevin- 1/utrophin complexes and the far greater abundance of β- dystrobrevin/Dp71 complexes in this tissue (Fig. 4). None of the proteins studied immunoprecipitated with the pre-immune sera (Fig. 4, lane 1), proving that these proteins are soluble and that their immunoprecipitation is specific. Fig. 3. Immunolocalisation of α-dystrobrevin-1, dystrophin, Dp71 and utrophin in adult mouse kidney. Mouse kidney cryosections were To determine the syntrophin content in the dystrobrevin labelled with the following antibodies: anti-α-dystrobrevin-1, α1CT- complexes in kidney and liver, protein complexes were FP (A,B); anti-utrophin, URD40 (C,D); anti-dystrophin, 2166 immunoprecipitated with antibodies specific to α-syntrophin (E,F); anti-dystrophin (∆C), 2401(G,H). Each antibody has a distinct (2688), β1-syntrophin (2689) and β2-syntrophin (2045) staining pattern. α1CT-FP labelled the glomerulus and blood vessels and analysed by western blotting. β-Dystrobrevin (A), and the smooth muscle of the renal pelvis (B, arrow). URD40 coimmunoprecipitated with all three antibodies in kidney and staining was detected in the glomerulus, the DCT (C) and the liver (Fig. 5A). α-Dystrobrevin-1, utrophin and Dp71∆C were collecting ducts (D, arrowhead). Weak staining of the large blood also found to coimmunoprecipitate with all three syntrophins vessels (D, inset) was also observed with this antibody. 2166 staining in kidney (Fig. 5B,C,E,kidney), whilst Dp71 could not be was absent from the renal cortex (E), but present in the smooth detected in any of the three immunoprecipitates due to the low muscle of the renal papilla (F, arrow) and the large blood vessels abundance of this protein in kidney (Fig. 5D,kidney). In liver, (F, inset). (G-H) 2401 stained renal tubules, Bowman’s capsules, the ∆ mesangium of the glomerulus (G), collecting ducts (H, arrowhead) the dystrobrevins, utrophin, Dp71 and Dp71 C predominantly and the large blood vessels (H, inset). bv, blood vessel; g, coimmunoprecipitated with β1-syntrophin, the most abundant glomerulus; t, cortical renal tubule. DCT, distal convoluted tubules. syntrophin isoform in liver (Fig. 5; Peters et al., 1997a). Bar, 100 µm. Utrophin and Dp71∆C also coimmunoprecipitated with liver Dystrobrevin complexes in kidney 2719

α- and β2-syntrophin, albeit to a much lesser extent (Fig. dystrobrevin-1 containing complexes in kidney, and five α- 5C,E,liver), whilst Dp71 coimmunoprecipitated with α- dystrobrevin-1 containing complexes in liver. syntrophin but not β2-syntrophin (Fig. 5D,liver). α- Dystrobrevin-1, by contrast, coimmunoprecipitated with β2- Localisation of the syntrophins in kidney syntrophin and not α-syntrophin (Fig. 5B,liver). Since the The localisations of α1-, β1- and β2-syntrophin were dystrobrevins and members of the dystrophin protein determined by immunofluorescence, using a panel of family contain syntrophin-binding sites, based on antibodies specific to each syntrophin isoform. α-Syntrophin coimmunoprecipitation data there are potentially 12 and 17 was detected at the basal membrane of cortical renal tubules different β-dystrobrevin containing complexes in kidney and (Fig. 6A), Bowman’s capsules (Fig. 6A), and collecting ducts liver, respectively. In addition there may be up to six α- (Fig. 6B), as well as blood vessels (Fig. 6B, inset). By contrast,

Fig. 4. Association of β-dystrobrevin and α-dystrobrevin-1 with utrophin and Dp71. Detergent-soluble extracts from kidney and liver were immunoprecipitated with preimmune sera (lane 1), β521 (lane 2), α1CT-FP (lane 3), URD40 (lane 4), 2166 (lane 5) and 2401 (lane 6). Coimmunoprecipitated proteins were analysed by western blotting with βCT-FP (A), α1CT-FP (B), URD40 (C), 2166 (D) and 2401 (E).β-Dystrobrevin coimmunoprecipitates with utrophin, Dp71 and Dp71∆C in kidney and liver, confirming that β-dystrobrevin is part of utrophin and Dp71 complexes in these tissues. By contrast, α-dystrobrevin-1 coprecipitates predominantly with utrophin in kidney and, to a much lesser extent, in liver. β-Dystrobrevin and α-dystrobrevin-1 do not copurify, implying they are part of separate complexes and are unlikely to associate with one another. Lane C is the loading control for each experiment, namely detergent-soluble extracts from kidney and liver; * indicates rabbit IgG heavy chain that is detected by the secondary antibody, and the arrows point to the coprecipitated proteins detected by the respective primary antibodies. 2720 N. Y. Loh and others

different kidney cell-types (Fig. 7). A complex comprising β- dystrobrevin, α- and β2-syntrophin and Dp71∆C is at the basal region of cortical renal tubules and Bowman’s capsules, whilst a complex comprising β-dystrobrevin, α- and β2- syntrophin and utrophin is at the basal region of collecting ducts. In addition, a β-dystrobrevin/β2-syntrophin/utrophin complex and an α-dystrobrevin-1/β2-syntrophin/utrophin complex are present at the glomerulus, whilst an α- dystrobrevin-1/β1-syntrophin/utrophin complex is found in peritubular capillaries, and an α-dystrobevin-1/α- syntrophin/β1-syntrophin/dystrophin complex is in vascular smooth muscle. Dystrobrevin-containing complexes are maintained even in the absence of dystrophin To determine the effect of the loss of Dp71, Dp71∆C and dystrophin on the assembly and localisation of the other proteins under investigation, the panel of antibodies against the dystrophin protein family, the dystrobrevins and the syntrophins was used to label kidneys of mice deficient for all dystrophin isoforms (mdx3Cv). As expected, no staining was detected with the anti-dystrophin antibodies 2401 and 2166 (Fig. 8A,A′ and B,B′). No difference was observed in the localisation of β-dystrobrevin (Fig. 8C,C′), α-dystrobrevin-1 (Fig. 8D,D′), utrophin (Fig. 8E,E′), α-syntrophin (Fig. 8F,F′) or β1-syntrophin (Fig. 8G,G′), implying that the localisation of these proteins is independent of Dp71, Dp71∆C and dystrophin. By contrast, there was a clear reduction of β2- syntrophin in the cortical renal tubules, Bowman’s capsule and glomeruli (Fig. 8H,H′), suggesting that anchoring of a fraction of β2-syntrophin to the basal membrane in these regions is dependent on the presence of Dp71∆C. To investigate if these proteins still formed a complex in the absence of Dp71∆C, coimmunoprecipitation experiments were performed on detergent extracts of mdx3Cv kidney with the panel of antibodies and detected with βCT-FP. β-Dystrobrevin Fig. 5. Coimmunoprecipitation of β-dystrobrevin, α-dystrobrevin-1, was found to coprecipitate with anti-utrophin URD40 and anti- utrophin and Dp71 with the syntrophins in kidney and liver. syntrophins 2688 (α), 2689 (β1) and 2045 (β2) (Fig. 8I). This Detergent-soluble extracts from kidney and liver were finding suggests that associations between β-dystrobrevin immunoprecipitated with antibodies specific to α-syntrophin (2688; and utrophin, α-, β1- and β2-syntrophin, respectively, are β β lane 1), 1-syntrophin (2689; lane 2) and 2-syntrophin (2045; lane unaffected by the absence of Dp71∆C. β-Dystrobrevin- 3). Coprecipitated proteins were detected by western blotting with β βCT-FP (A), α1CT-FP (B), URD40 (C), 2166 (D) and 2401 (E). In detection in the anti- 2-syntrophin lane can be explained by β α the presence of β2-syntrophin containing complexes in kidney extracts, -dystrobrevin, -dystrobrevin-1, utrophin and β Dp71∆C coprecipitate with all three syntrophins, whilst in liver collecting ducts since the localisation of the 2-syntrophin in extracts, all five proteins analysed predominantly coprecipitate with this region is unchanged (data not shown). β1-syntrophin. In addition, β-dystrobrevin, utrophin, Dp71 and In summary, we have shown that the different dystrobrevin Dp71∆C coprecipitate with liver α-syntrophin, whilst β- containing protein complexes are present in different regions dystrobrevin, α-dystrobrevin-1, utrophin and Dp71∆C coprecipitate of the kidney. Furthermore, we have shown evidence that with liver β2-syntrophin, albeit to a much lesser degree. The loading Dp71∆C is unnecessary for the correct localisation and control for each experiment (lane C) contains detergent-soluble association of β-dystrobrevin and α-syntrophin at the basal extracts from kidney and liver. * marks the rabbit IgG heavy chain. membrane of cortical renal tubules. the presence of β1-syntrophin was observed only in peritubular DISCUSSION capillaries (Fig. 6C), major blood vessels (Fig. 6D, inset), and the smooth muscle of the renal pelvis (Fig. 6D). β2-syntrophin In this paper we have determined the expression, distribution, was detected in the same regions as α-syntrophin (Fig. 6E,F). and organisation of protein complexes containing members of It was also observed in glomeruli (Fig. 6E). the dystrophin, dystrobrevin and syntrophin protein families in Thus, based on immunohistochemical and normal and dystrophin-deficient mouse kidneys. Hitherto, coimmunoprecipitation data, we have identified at least six studies of these proteins have been carried out largely in different dystrobrevin-containing complexes present in muscle and brain. To our knowledge, detailed study of similar Dystrobrevin complexes in kidney 2721

Fig. 6. Differential localisation of the syntrophins in kidney. Serial sections of adult mouse kidney were labelled with the anti-α-syntrophin antibody 2688 (A,B), anti-β1-syntrophin antibody 2689 (C,D) and anti-β2-syntrophin antibody 2045 (E,F). α- and β2-syntrophin are present at the renal tubules, collecting ducts (arrowheads) and Bowman’s capsule (A,B,E,F). In addition, β2-syntrophin is present at the glomerulus (E). By contrast, β1-syntrophin is restricted to peritubular capillaries (C) and smooth muscle cells of blood vessels (D, inset) and the renal pelvis (D, arrow). Insets, blood vessel; g, glomerulus; t, cortical renal tubule. Bar, 100 µm.

kidney complexes is limited to a single protein complex in a functions of the constituent proteins have yet to be fully single cell culture system, namely a utrophin-associated established, there is evidence to show that the dystrophin-like protein complex in the Madin-Darby canine kidney (MDCK) complexes act as scaffolds for signalling molecules. As cell-line (Kachinsky et al., 1999). The present study shows that mentioned previously, the syntrophins associate with a the story in the renal system is more complicated than that variety of signalling molecules via their PDZ domains observed in the in vitro system. We find that α-dystrobrevin-1 (Brenman et al., 1996; Gee et al., 1998; Lumeng et al., 1999). and β-dystrobrevin, the two members of the dystrobrevin More importantly, there is evidence for a differential family expressed in kidney, are associated with different association between the syntrophins and their different members of the dystrophin and syntrophin family of proteins binding partners. For example MAST205 has been shown to to form different complexes in renal epithelial, endothelial and associate with β2-syntrophin but not α-syntrophin (Lumeng smooth muscle cells. Furthermore, we show that, unlike in et al., 1999). Likewise, muscle-expressed voltage-gated skeletal muscle where dystrophin is the vital for the assembly sodium channels show higher affinity for α- and β2- and maintenance of the DPC, the absence of dystrophin and its syntrophin than for β1-syntrophin (Gee et al., 1998). Thus short isoforms have relatively little effect on the localisation different combinations of syntrophins would determine the and assembly of the dystrobrevins and syntrophins in these proteins that associate with different dystrobrevin complexes. cells. Of the known interacters, nNOS and MAST205 are expressed in kidney (Tojo et al., 1997; Wang et al., 1998; Lumeng et Functional significance of different dystrobrevin al., 1999). nNOS is involved in regulating renal function complexes (Bachman and Mundel, 1994; Lu et al., 1997; Ichira et al., Our studies have provided fresh insights into the kidney 1998). MAST205 was first identified as a microtubule- dystrobrevin complexes. Firstly, the existence of at least six associated protein in mouse testis, and plays a role in dystrobrevin complexes localised to different regions of the spermatid maturation (Walden and Cowan, 1993; Walden and kidney (Fig. 7) implies that there are likely to be differences Millette, 1996). Therefore, it is likely that the renal in their functions in the different renal cell-types. Whilst the dystrobrevin complexes are involved in kidney function and 2722 N. Y. Loh and others

DCT Dp71∆C, β-db, Arterioles α-syn, β2-syn dys, α-db1, Collecting α-syn, β1-syn tubule Dp71∆C, utro, β-db, α-syn, β1-syn, β2-syn

Mesangium Dp71∆C, β-db, β2-syn

Collecting duct utro, β-db, PCT α-syn, β2-syn Dp71∆C, β-db, Glomerular α β -syn, 2-syn capillary utro, α-db1, Podocytes β2-syn utro, β-db, β2-syn Arcuate artery dys, α-db1, α-syn, β1-syn

Fig. 7. Schematic representation of the nephron showing the localisation of the various dystrobrevin complexes. Dystrobrevin complexes listed here are based on immunoprecipitation and immunolocalisation data. dys, dystrophin; utro, utrophin; β-db, β-dystrobrevin; α-db1, α-dystrobrevin- 1; α-syn, α-syntrophin; β1-syn, β1-syntrophin; β2-syn, β2-syntrophin. PCT, proximal convoluted tubules; DCT, distal convoluted tubules. development by acting as scaffolds for these and other so far to be the case in kidney (Fig. 8). In dystrophin-deficient unidentified signalling molecules. kidneys the localisation of and interaction between β- dystrobrevin and α-syntrophin at the basal region of cortical Is β-dystrobrevin a key regulator of complex renal tubules are unaffected in the absence of Dp71∆C (Fig. assembly in kidney epithelia? 8C′,E′,F′,I). Furthermore, the retention of these proteins is not Our studies also provide further insight into the assembly of due to any significant increase in levels of utrophin in this the kidney dystrobrevin complexes. The kidney dystrobrevin region (Fig. 8E,E′). Likewise, localisation of the dystrobrevins complexes lie in close juxtaposition with glomerular and and syntrophins were largely unaffected in kidneys of tubular basement membranes where dystroglycan is known to utrophin-null mice (utrn−/−) and those of utrn−/−;mdx3Cv be present (Durbeej et al., 1998). Although it has not been mice (N. Y. Loh et al., manuscript in preparation). Similar proved, it is likely that dystroglycan is the transmembrane link findings have been reported of dystrophin-like complexes in between various dystrobrevin complexes and renal basement brain (Blake et al., 1999). Our findings suggest that another membranes. However, whilst the dystrophin-dystroglycan link molecule, perhaps β-dystrobrevin, is involved in the assembly is necessary for the assembly and localisation of the and maintenance of the kidney complexes. In addition, the syntrophins and dystrobrevins in muscle, this does not appear constituent proteins of these complexes may not be as tightly Dystrobrevin complexes in kidney 2723

Fig. 8. Dystrobrevins, utrophin and syntrophins in mdx3cv kidneys. Serial sections of normal (A-H) and mdx3cv (A′-H′) kidneys were labelled with the following antibodies: anti-dystrophin antibodies 2401 (A,A′), and 2166 (B,B′); anti-β-dystrobrevin β521 (C,C′); anti-α-dystrobrevin-1 α1CT-FP (D,D′); anti-utrophin URD40 (E,E′); anti-α-syntrophin 2688 (F,F′); anti-β1-syntrophin 2689 (G,G′); anti-β2-syntrophin 2045 (H,H′). No difference was observed in the localisation of the dystrobrevins, utrophin, α1-syntrophin or β1-syntrophin in the absence of Dp71 and dystrophin. By contrast a dramatic reduction of labelling intensity of β2-syntrophin staining was observed in cortical renal tubules and the Bowman’s capsule, which is in agreement with the notion that β2-syntrophin directly binds to Dp71∆C in these regions. Bar, 100 µm. (I) Detection of β-dystrobrevin (β) in protein complexes from mdx3cv kidneys immunoprecipitated with preimmune sera (lane 1), β521 (lane 2), α1CT-FP (lane 3), URD40 (lane 4), 2166 (lane 5), 2401 (lane 6), 2688 (lane 7), 2689 (lane 8) and 2045 (lane 9). * denotes IgG heavy chain. An arrowhead marks a breakdown product of β-dystrobrevin. β-dystrobrevin-syntrophin interactions are still formed, implying that Dp71 and dystrophin are not necessary for β-dystrobrevin-syntrophin associations. associated as the muscle DPC. The latter notion is consistent independent of members of the dystrophin protein family. with findings of Durbeej and Campbell (1999) on the Further understanding of the roles of kidney complexes may utrophin-dystroglycan-ε- complex in epithelia of lie in the identification of binding partners of the syntrophins kidney and liver. This difference in the effect of dystrophin- and dystrobrevins. deficiency on kidney and muscle complexes may help explain the lack of kidney pathology in mdx3Cv mice and DMD This work was generously supported by grants from the Wellcome patients. Trust. N.Y.L was a Rhodes Scholar. D.J.B. is a Wellcome Trust In summary, we have identified at least six different Career Development Fellow. S.E.N. is a Wellcome Trust Prize dystrobrevin-containing complexes that are present in different Student. The authors wish to thank Allyson Potter and Nicholas cell types in the kidney. Moreover, we have shown that Owen for their expert technical assistance, and Dr Albert Ong for assembly and localisation of these complexes are largely helpful discussions. 2724 N. Y. Loh and others

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