Cloning of two novel ADP-ribosylation factor-like proteins and characterization of their differential expression in 3T3-L1 cells.
A polymerase chain reaction-based cloning approach was employed in order to identify ADP-ribosylation factors (ARF) in murine 3T3-L1 cells and to study their expression before and after differentiation of cells to the adipocyte-like phenotype. Partial sequences comprising the effector domains of ARF were amplified with degenerate primers and cloned. Five of these sequences were identified as murine homologues of known human ADP-ribosylation factors (ARF 1, 2, 4, 5, and 6). In addition, partial sequences of two previously unknown isoforms were found, and complete cDNA clones were isolated from a rat fat cell library and were sequenced. Both sequences harbor a putative myristoylation site in position 2, the known consensus sequences presumably involved in GTP binding and hydrolysis, and lack cysteine residues in the C terminus. Their amino acid sequences share a 56 and 41% identity, respectively, with human ARF 1. Based on a comparison with the known ARF isoforms, the first clone appears to represent the mammalian homologue of a known sequence from Drosophila (dARL 1, 79% identity) and was therefore designated rARL 1. The second clone resembled none of the known ARF-like proteins and was designated rARL 4. mRNA of ARL 4 was undetectable in the fibroblasts but abundant in the adipocyte-like phenotype, its expression starting on day 6 of the differentiation. In contrast, ARF 1, 2, and 5 were unaltered by differentiation of the 3T3-L1 cells; mRNA levels of ARF 6, and also of ARL 1 and ARF 4, were reduced after differentiation. It is suggested that the function of ARL 4 is related to the adipocyte-like phenotype of 3T3-L1 cells.
Schürmann
A
A
Institut für Pharmakologie und Toxikologie der RWTH Aachen, Germany.
Breiner
M
M
Becker
W
W
Huppertz
C
C
Kainulainen
H
H
Kentrup
H
H
Joost
H G
HG
D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D017667:Adipocytes / Q000378:metabolism*; D000273:Adipose Tissue / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003062:Codon; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000096:biosynthesis* / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D051381:Rats; D012016:Reference Values; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D000089982:ADP-Ribosylation Factor 6; D003062:Codon; D018076:DNA, Complementary; D009227:Myristic Acids; D019814:Myristic Acid; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C486431:Arl4a protein, mouse; C486430:Arl4a protein, rat; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C116036:ARL4A protein, human; C000715727:Arf6 protein, mouse; C000715349:Arf6 protein, rat