SPLICING ISOFORMS OF RAT ASH GRB2 - ISOLATION AND CHARACTERIZATION OFTHE 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) c DNA from rats. One of these clones, Ash-psi was an unusual transcribed gene having 93% identity in the nucleotide sequence to Ash-l. The oth er two clones, Ash-m and -s, had nucleotide sequences identical with A sh-l cDNA in the amino-terminal region. The coding sequence of Ash-m c DNA is 42 nucleotides shorter than that of Ash-l cDNA. The defective r egion 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 se quence, thereby differing from Ash-l cDNA Cloning of the genomic DNA o f 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 intro ns. Ash-m and Ash-s mRNAs were detected in various tissues by reverse- transcribed polymerase chain reaction. Ash-m physically associated wit h dynamin, but the association with Sos was less effective than that o f Ash-l in rat pheochromocytoma PC12 cell lysates, irrespective of tre atment with nerve growth factor. In contrast, Ash-s formed a complex w ith dynamin and Sos in cell lysates. Moreover, the newly formed carbox yl-terminal SH3 of Ash-m by splicing bound different proteins from tho se bound to the carboxyl-terminal SH3 domain of Ash-l, suggesting that Ash-m generates different signals. Microinjection of Ash-m or Ash-s i nto Balb/c 3T3 cells inhibited DNA synthesis induced by platelet-deriv ed growth factor. These results show that these isoforms act as domina nt negative regulators of mitogenic signals by Ash-l.