Splicing isoforms of rat Ash/Grb2. Isolation and characterization of the cDNA and genomic DNA clones and implications for the physiological roles of the isoforms.
We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-psi, was an unusual transcribed gene having 93% identity in the nucleotide sequence to Ash-l. The other two clones, Ash-m and -s, had nucleotide sequences identical with Ash-l cDNA in the amino-terminal region. The coding sequence of Ash-m cDNA is 42 nucleotides shorter than that of Ash-l cDNA. The defective region of Ash-m cDNA encodes 14 amino acid residues (157 to 170 of Ash-l), which comprise the most conserved region of the second SH3 domain. On the other hand, the coding sequence of Ash-s terminated at the end of the first SH3 domain due to a stop codon at the boundary of the sequence, thereby differing from Ash-l cDNA. Cloning of the genomic DNA of the Ash-l-encoding gene, determination of the gene organization, and nucleotide sequencing revealed that the two isoforms, as well as Ash-l, are generated from a single gene by unusual alternative splicings. The gene spans more than 16 kilobases and contains 6 exons and 5 introns. Ash-m and Ash-s mRNAs were detected in various tissues by reverse-transcribed polymerase chain reaction. Ash-m physically associated with dynamin, but the association with Sos was less effective than that of Ash-l in rat pheochromocytoma PC12 cell lysates, irrespective of treatment with nerve growth factor. In contrast, Ash-s formed a complex with dynamin and Sos in cell lysates. Moreover, the newly formed carboxyl-terminal SH3 of Ash-m by splicing bound different proteins from those bound to the carboxyl-terminal SH3 domain of Ash-l, suggesting that Ash-m generates different signals. Microinjection of Ash-m or Ash-s into Balb/c 3T3 cells inhibited DNA synthesis induced by platelet-derived growth factor. These results show that these isoforms act as dominant negative regulators of mitogenic signals by Ash-l.
Watanabe
K
K
Department of Experimental Biology, Tokyo Metropolitan Institute of Gerontology, Japan.
Fukuchi
T
T
Hosoya
H
H
Shirasawa
T
T
Matuoka
K
K
Miki
H
H
Takenawa
T
T
D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D018076:DNA, Complementary / Q000302:isolation & purification*; D051380:GRB2 Adaptor Protein; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011506:Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D051381:Rats
D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; D051380:GRB2 Adaptor Protein; C495950:Grb2 protein, rat; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D004247:DNA