Conversion of G-protein specificity of insulin-like growth factor II/mannose 6-phosphate receptor by exchanging of a short region with beta-adrenergic receptor.
The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.
Takahashi
K
K
Department of Medicine, Harvard Medical School, Massachusetts General Hospital-East, Charlestown 02129.
Murayama
Y
Y
Okamoto
T
T
Yokota
T
T
Ikezu
T
T
Takahashi
S
S
Giambarella
U
U
Ogata
E
E
Nishimoto
I
I
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D017527:Receptor, IGF Type 2 / Q000096:biosynthesis / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D017527:Receptor, IGF Type 2; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases