Ciated LIM ProteinFigure 2. Sequence analysis of ALP isoforms. (A) Amino acids 50 of ALP

Ciated LIM ProteinFigure 2. Sequence analysis of ALP isoforms. (A) Amino acids 50 of ALP encode a consensus PDZ domain. Alignment of ALP with PDZ domains from CLP-36, PSD95, 1-syntrophin, ( 1syn), nNOS, and INAD. Histidine 62 of ALP is marked with an asterisk and leucine 78 with a pound sign. (B) Predicted sequences of rat ALP (GenBankEMBLDDBJ accession no. AF002281) and human ALP (hALP) are aligned with two homologous proteins, CLP-36 and RIL. (C) An option ALP isoform is expressed in the heart. Schematic model shows the domain structure of ALP along with the Ach Inhibitors Related Products divergence of ALP amongst skeletal muscle (hALPSK; accession no. AF002280) and heart (hALPH; accession no. AF002282). The alignment shows that the central region of ALP is different among skeletal muscle and heart. The accession numbers for ESTs utilized to construct hALPH are F12229, R20192, AA147575, AA211287, and D56502. The accession numbers for ESTs encoding the skeletal muscle pecific splice for hALPsk are Z28845, Z19288, and Z28703.The central region of ALP showed no homology to any other cloned gene although the COOH terminus encodes a LIM domain. Although ALP has not previously been reported, other proteins with a similar domain structure have already been described. A database homology search with BLAST indicated that ALP shares higher homology to quite a few newly identified transcripts which includes CLP-36, RIL, and enigma (Fig. two B). CLP-36 was identified as a cDNA whoseexpression AGK Inhibitors MedChemExpress within the heart is downregulated by hypoxia (Wang et al., 1995). RIL, quick for reversion-induced LIM protein, is downregulated in H-ras ransformed cells (Kiess et al., 1995). Enigma was identified as an insulin receptor nteracting protein (Wu et al., 1996). These investigators, nonetheless, didn’t recognize the homology from the NH2-terminal regions of CLP-36, RIL, or enigma using the PDZ domain. The PDZ domain of ALP shares 55, 48, and 45 aminoThe Journal of Cell Biology, Volume 139,acid identity with the PDZ domains of CLP36, RIL, and enigma, respectively. The LIM domain of ALP shares even stronger homology (67 identity) with CLP36 and RIL. Whilst ALP, CLP36, and RIL all only have one LIM domain, enigma has three LIM domains. The sequence homology indicates that ALP, CLP36, and RIL constitute a new household of proteins containing an NH2-terminal PDZ domain as well as a COOH-terminal LIM domain. Our evaluation with the expressed sequence tag (EST) database showed that overlapping cDNAs corresponding to human ALP have already been deposited. The human ALP is 91 identical for the rat sequence. We noted that EST clones from human heart libraries were consistently unique inside the central area from those in human skeletal muscle libraries (Fig. 2 C). Exons encoding the central 112 amino acids of skeletal muscle ALP are most likely to become spliced out within the heart and replaced by exons encoding 64 diverse amino acids. To confirm this differential expression, we amplified the region that was distinctive to heart transcripts and reprobed the Northern blot. As anticipated, we identified heart-specific expression of this region of ALP (data not shown). We as a result define two subtypes ALPSK and ALPH for the option transcripts that occur in skeletal muscle and heart, respectively.The PDZ Domain of ALP Binds -Actinin-Previous studies have shown that PDZ domains participate in protein rotein interactions. To identify possible targets for the PDZ domain of ALP, we used the yeast two-hybrid method. We screened 106 clones from an adult skeletal muscle library (Clo.