Cloning of the rat ErbB3 cDNA and characterization of the recombinant protein.
Three cDNA fragments that encoded all but the extreme N terminus of the rat ErbB3 protein were cloned by low-stringency screening of a rat liver cDNA library with a human ERBB3 probe. The remaining 5'-end of the cDNA was generated by a reverse transcription-polymerase chain reaction method, and a single full-length rat ErbB3 cDNA was assembled. A comparison of the deduced amino acid (aa) sequences of human and rat ErbB3 was made, and the effects of certain aa substitutions in the putative protein tyrosine kinase domain were considered. The rat ErbB3 cDNA was subsequently expressed in cultured NIH-3T3 mouse fibroblasts, in which a high level of approx. 180-kDa recombinant ErbB3 (re-ErbB3) was generated. The rat re-ErbB3 produced in transfected fibroblasts was responsive to the polypeptide, heregulin, a known ligand for ErbB3. Challenge of transfected fibroblasts with heregulin stimulated the phosphorylation of rat re-ErbB3 on Tyr residues and promoted its association with the p85 subunit of phosphatidylinositol 3-kinase. Together, these results indicate that a fully functional rat ErbB3 cDNA has been isolated, and that fibroblast cells expressing this cDNA will be suitable for investigations of the signal transduction mechanism of ErbB3.
Hellyer
N J
NJ
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Kim
H H
HH
Greaves
C H
CH
Sierke
S L
SL
Koland
J G
JG
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000502:physiology; D006801:Humans; D008099:Liver / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020890:Neuregulin-1; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D020893:Receptor, ErbB-3; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics
D002352:Carrier Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D020890:Neuregulin-1; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C092262:heregulin beta 1 (177-244); D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D066246:ErbB Receptors; D020893:Receptor, ErbB-3