Eur. J. Biochem. 235, 103-113 (1996) zyxwvutsr 0 FEBS 1996 zyxwvutsrqpo A novel dystrophidutrophin-associated protein is an enzymatically inactive member of the phosphoglucomutase superfamily Elena P. MOlSEEVA', Alexey M. BELKIN', Nigel K. SPURR?, Victor E. KOTELIANSKY4 and David R. CRITCHLEY' I Department zyxwvutsrq of Biochemistry, University of Leicester, UK zyxwvutsrq ' Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, UK Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, North Carolina, USA Laboratoire de Physiopathologie du Developpement, CNRS et Ecole Normal Superieure, Paris, France (Received 4 September/3 Novcmber 1995) - EJB 95 1453/1 A 60-kDa protein localised in adherens-type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin IBelkin, A. M. & Burridge, K. (1995) .I. zyx Biol. Chern. 270, 6328-63371. In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins. The PGM- related protein (PGM-RP), which contains 506 amino acids (55.6 kDa), is smaller than PGMl (566 amino acids, 61 kDa). The active site consensus sequences of prokaryotic and eukaryotic mutases are not con- served in PGM-RP, a finding consistent with the lack of enzymatic activity of PGM-RP in vitvo, and the absence of a phosphorylated intermediate in vivo. The organisation of the PGM-RP gene is essentially identical to that of PGMl. We propose that the PGM-RP gene, which we have mapped to human chromo- some 9qcen-ql3, evolved from the PGMl gene, and encodes a protein with a structural rather than an enzymatic role. PGM-RP is expressed predominantly in muscle with the highest levels in smooth muscle. The significance of the interaction between dystrophinhtrophin and an increasing number of cytoplasmic proteins including PGM-RP remains to be explored. Keywords: phosphoglucomutase ; adherens-type cellular junctions ; dystrophin ; utrophin. The gene mutated in the X-linked Duchenne and Becker- type muscular dystrophies encodes a 427-kDa protein (dystrophin) that is homologous to the a-actinin and speclrin family of cytoskeletal proteins 11, 21. Dystrophin is an elongated inolecule 13 I which contains an N-terminal actin-binding domain 141, a central rod-like domain containing 25 spectrin-like repeats, a cysteine-rich domain containing two incomplete EF-hand calcium-binding motifs, and a C-terminal region containing a possible leucine zipper [5]. It is localised on the cytoplasmic face of the sarcolemma where it may serve to protect the sarco- lemma from the mechanical stresses developed during muscle contraction 16, 71. Some information on the nature of the link between dystrophin and the membrane has recently been ob- tained, although the observation that the DMD gene gives rise to multiple dystrophin isoforins suggests that current models will require modification [S]. The C-terminal region of dystrophin is thought to contain binding sites for a complex of five trans- membrane proteins (adhalin, P-dystroglycan, 43-kDa DAG (dystrophin-associated glycoprotein), 35-kDa DAG and 25-kDa DAP (dystrophin-associated protein) four of which are in turn Correspondence to D. R. Critchley, Department of Biochemistry, University of Leicester, University Rd, Leicester LEI 7RH, UK zyxwvuts Fax: +44 116 2523369. Ahhreviutions. PGM, phosphoglucomutase; PGM-RP, phosphoglu- coniulase-relatcd protein; DAG, dystropbin-associated glycoprotein; DAP, dystrophin-associated protein. Ennzynze. Phosphoglucomutase (EC 5.4.2.2). Note. The PGM-RP cDNA sequence data have been deposited with the GSDB/EMBL/NCBI sequence data banks and are available under acccssion number L40933. The sequences of exoiis 2, zyxwvuts 3,S, 8 and 9 of the muse PGM-KP gene are available under accession numbers L42902- L42905 and L42907, respectively. linked to a large extracellular glycoprotein a-dystroglycan 12, 81. This latter protein binds to the laminin isoform merosin, provid- ing a link between the extracellular matrix and the muscle cy- toskeleton. At least three additional cytoplasmic proteins (al-, [jl-, and p2-syntrophins) have been identified that bind to the C- terminal region of dystrophin, although their function is un- known [2, 8, 91. al-Syntrophin and PI-syntrophin have been shown to bind to the alternatively spliced dystrophin exons 73 and 74, respectively 19, 101. The dystrophin-related protein, utrophin 121, has also been shown to interact with some of the same dystrophin-associated proteins [l I]. Utrophin is expressed in embryonic and regenerating muscle where it is localised to the sarcolemma, but it is replaced by dystrophin in adult skeletal muscle 121. However, it is expressed in many different cell types with high levels of expression in smooth muscle. Down-regula- tion of utrophin in cultured astroctyes using antisense oligonu- cleotides, leads to a marked reduction in cell-matrix contacts which implies that the utrophin cytoskeletal complex is also in- volved in cell adhesion [12]. In an attempt to identify additional muscle proteins localised at the membranekytoskeletal interface, we raised a panel of mo- noclonal antibodies to a human uterine smooth muscle cytoskel- eta1 extract. We identified a novel antigen (60-63 kDa) which was localised in a variety of adherens-type cellular junctions [I 3 1. This protein, initially called aciculin 11 41, has recently been shown to coimmunoprecipitate with dystrophin and utrophin [15]. In this study, we report the nucleotide and deduced amino acid sequence of this protein which turns out to be a novel men- ber of the phosphoglucomutase (PGM) family of proteins. Since the protein lacks phosphoglucomutase activity, we refer to the protein as the phosphoglucomutase-related protein or PGM-RP.