Identification of two isoforms of mouse neuropeptide Y-Y1 receptor generated by alternative splicing. Isolation, genomic structure, and functional expression of the receptors.
Two cDNA clones homologous with human neuropeptide (NP) Y-Y1 receptor have been isolated from a mouse bone marrow cDNA library. One was thought to be the cognate of the human NPY-Y1 receptor, termed Y1 alpha receptor, and the other form, termed Y1 beta receptor, differed from the Y1 alpha receptor in the seventh transmembrane domain and C-terminal tail. Analysis of the mouse genomic DNA showed that both receptors originated from a single gene. The different peptide sequences of the Y1 beta receptor were encoded by separate exons, hence, these receptors were generated by differential RNA splicing. High affinity binding of [125I]NPY to each receptor expressed in Chinese hamster ovary (CHO) cells and sequestration of [125I]NPY after binding to each receptor were observed. In the CHO cells expressing the Y1 alpha receptor, intracellular Ca2+ increase, inhibition of forskolin-induced cAMP accumulation, and mitogen-activated protein kinase (MAPK) activation were observed by stimulation of NPY, and these responses were abolished by pretreatment with pertussis toxin. Since wortmannin completely inhibited NPY-elicited MAPK activation, we speculate that wortmannin-sensitive signaling molecule(s) such as phosphoinositide 3-kinase may lie between pertussis toxin-sensitive G-protein and MAPK. In contrast, these intracellular signals were not detected in CHO cells expressing the Y1 beta receptor. Northern blots and reverse transcriptase-polymerase chain reaction analyses indicated that the Y1 alpha receptor was highly expressed in the brain, heart, kidney, spleen, skeletal muscle, and lung, whereas the Y1 beta receptor mRNA was not detected in these tissues. However, the Y1 beta receptor was expressed in mouse embryonic developmental stage (7 and 11 days), bone marrow cells and several hematopoietic cell lines. These results suggest that the Y1 beta receptor is an embryonic and a bone marrow form of the NPY-Y1 receptor, which decreases in the expression during development and differentiation.
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa, Japan.
M
M
Nakamura
C
C
Sakanaka
Y
Y
Aoki
H
H
Ogasawara
T
T
Tsuji
H
H
Kodama
T
T
Matsumoto
T
T
Shimizu
M
M
Noma
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001853:Bone Marrow / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007438:Introns; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000378:metabolism* / Q000494:pharmacology; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D002614:Chelating Agents; D017931:DNA Primers; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C097212:neuropeptide Y-Y1 receptor; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D037342:Pertussis Toxin; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium; D000077191:Wortmannin