RESUMO
Members of the cysteine-rich protein (CRP) family are evolutionarily conserved proteins that have been implicated in the processes of cell proliferation and differentiation. In particular, one CRP family member has been shown to be an essential regulator of cardiac and skeletal muscle development. Each of the three vertebrate CRP isoforms characterized to date is composed of two copies of the zinc-binding LIM domain with associated glycine-rich repeats. In this study, we have addressed the biological significance of the CRP multigene family by comparing the subcellular distributions, biochemical properties, and expression patterns of CRP1, CRP2, and CRP3/MLP. Our data reveal that all three CRP family members, when expressed in adherent fibroblasts, associate specifically with the actin cytoskeleton. Moreover, all three CRP isoforms are capable of interacting with the cytoskeletal proteins alpha-actinin and zyxin. Together, these observations suggest that CRP family members may exhibit overlapping cellular functions. Differences between the three CRPs are evident in their protein expression patterns in chick embryos. CRP1 expression is detected in a variety of organs enriched in smooth muscle. CRP2 is restricted to arteries and fibroblasts. CRP3/MLP is dominant in organs enriched in striated muscle. CRP isoform expression is also developmentally regulated in the chick. Our findings suggest that the three CRP family members perform similar functions in different muscle derivatives. The demonstration that all members of the CRP family are associated with cytoskeletal components that have been implicated in the assembly and organization of filamentous actin suggests that CRPs contribute to muscle cell differentiation via effects on cytoarchitecture.
Assuntos
Cisteína , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica , Variação Genética , Zíper de Leucina , Proteínas Nucleares/química , Fatores de Transcrição/química , Actinas/análise , Sequência de Aminoácidos , Animais , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT , Embrião de Galinha , Galinhas , Sequência Conservada , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Fibroblastos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/biossíntese , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
Here we describe a family of closely related LIM domain proteins in avian cells. The LIM motif defines a zinc-binding domain that is found in a variety of transcriptional regulators, proto-oncogene products, and proteins associated with sites of cell-substratum contact. One type of LIM-domain protein, called the cysteine-rich protein (CRP), is characterized by the presence of two LIM domains linked to short glycine-rich repeats and a potential nuclear localization signal. We have identified and characterized two evolutionarily conserved members of the CRP family, CRP1 and CRP2, in chicken and quail. Expression of the genes encoding both CRP1 and CRP2 is differentially regulated in normal versus transformed cells, raising the possibility that members of the CRP family may function in control of cell growth and differentiation.
Assuntos
Proteínas Aviárias , Proteínas Musculares/genética , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Embrião de Galinha , DNA Complementar/química , Fibroblastos/metabolismo , Dados de Sequência Molecular , Família Multigênica , Proteínas Musculares/química , Proteínas/química , Proteínas Proto-Oncogênicas c-myc/química , Codorniz/embriologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.