ABSTRACT
STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.
Subject(s)
Genome, Human , Neoplasm Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/genetics , Cell Adhesion Molecules , Chromosome Mapping , Codon, Initiator , Drosophila melanogaster/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/isolation & purification , Neoplasm Proteins/metabolism , Protein Biosynthesis , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Stromal Interaction Molecule 1 , Stromal Interaction Molecule 2ABSTRACT
STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.
Subject(s)
Membrane Proteins , Neoplasm Proteins/metabolism , Phosphoproteins/metabolism , Amino Acid Sequence , Antibodies/chemistry , Biotinylation , Cell Line , Cell Membrane/metabolism , Fluorescent Antibody Technique , Glycosylation , Humans , Immunoblotting , Marine Toxins , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/immunology , Oxazoles/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation/drug effects , Precipitin Tests , Protein Processing, Post-Translational , Stromal Interaction Molecule 1 , Tumor Cells, CulturedSubject(s)
Iodohippuric Acid , Kidney Diseases/diagnosis , Radioisotope Renography/methods , Adolescent , Female , Functional Laterality , Humans , MaleSubject(s)
Urinary Tract Infections , Urologic Diseases , Female , Humans , Male , Urinary Catheterization , Urination DisordersABSTRACT
It is generally believed that after an intravenous injection of Hippuran its concentration in the plasma can be described as the sum of two exponentials. However, by collecting samples during the first minute after injection of the tracer, a third exponential term was found with a half-life of less than one minute. To explain its presence it was assumed that the plasma shares its Hippuran with two peripheral compartments, the rate of transfer from one compartment to another being proportional to the amount in the first of them. The proportionality factors have been determined for 20 adults and averaged. Substituting these averages in the differential equations for the distribution process, the only variable remaining is the renal excretion rate a (the fraction of plasma cleared per minute by the kidneys). If this procedure, which appears to be justified by the experimental results, is correct the shape of the plasma curve will be determined exclusively by a. It is shown that the (small) third compartment has a minor influence on the shape of the renogram curves.
