The neuropeptide Y Y5 receptor gene generates two splice variants, referred to here as Y5L (long isoform) and Y5S (short isoform). Y5L mRNA differs from Y5S mRNA in its 5′ end, generating a putative open reading frame with 30 additional nucleotides upstream of the initiator AUG compared with the Y5S mRNA. The purpose of the present work was to investigate the existence of the Y5L mRNA. The authenticity of this transcript was confirmed by isolating part of its 5′ untranslated region through 5′ rapid amplification of cDNA ends and analysing its tissue distribution. To study the initiation of translation on Y5L mRNA, we cloned the Y5L cDNA and two Y5L cDNA mutants lacking the first or the second putative initiation start codon. Transient expression of the three plasmids in COS-7 cells and saturation binding experiments using 125I-labelled polypeptide YY (PYY) as a ligand showed that initiation of translation on Y5L mRNA could start at the first AUG, giving rise to a Y5L receptor with an N-terminal 10-amino-acid extension when compared with the Y5S receptor. The human Y5L and Y5S receptor isoforms displayed similar affinity constants (1.3nM and 1.5nM respectively). [125I]PYY binding to COS-7 cells expressing either the Y5L or the Y5S isoform was inhibited with the same rank order of potency by a selection of six chemically diverse compounds: PYY>neuropeptide Y>pancreatic polypeptide>CGP71683A>Synaptic 34>Banyu 6. Comparison of the tissue distribution of Y5L and Y5S mRNAs, as determined by reverse transcription—PCR analysis, indicated that expression of Y5L mRNA occurs in a tissue-specific manner. Finally, we have shown that the two AUG triplets contained in the 5′ untranslated region of Y5L mRNA did not affect receptor expression.
- initiation codon context
- NPY receptor
- 5′ untranslated region
↵1 Present address: Sanofi-Synthelabo Recherche, Departement Cardiovasculaire Thrombose 1, Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France.
↵2 Present address: Aventis Pharma S.A., Centre de Recherche de Paris Etablissement de Vitry 13, quai Jules Guesdes, F 94400 Vitry-sur-Seine, France.
↵3 Present address: Biogema, 24 Avenue des Landais, 63170 Aubière, France.
↵4 Present address: EntoMed SA, Rue Tobias Stimmer, 67400 Illkirch, France.
Abbreviations used: CRE, cAMP response element; MSH, melanocyte-stimulating hormone; NPY, neuropeptide Y; PP, pancreatic polypeptide; PYY, polypeptide YY; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription—PCR; UTR, untranslated region; Y5L and Y5S, long and short isoforms respectively of the neuropeptide Y Y5 receptor.
- The Biochemical Society, London ©2003