RESUMO
The neuropeptide Y Y5 receptor gene generates two splice variants, referred to here as Y5(L) (long isoform) and Y5(S) (short isoform). Y5(L) mRNA differs from Y5(S) mRNA in its 5' end, generating a putative open reading frame with 30 additional nucleotides upstream of the initiator AUG compared with the Y5(S) mRNA. The purpose of the present work was to investigate the existence of the Y5(L) 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 Y5(L) mRNA, we cloned the Y5(L) cDNA and two Y5(L) 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 (125)I-labelled polypeptide YY (PYY) as a ligand showed that initiation of translation on Y5(L) mRNA could start at the first AUG, giving rise to a Y5(L) receptor with an N-terminal 10-amino-acid extension when compared with the Y5(S) receptor. The human Y5(L) and Y5(S) receptor isoforms displayed similar affinity constants (1.3 nM and 1.5 nM respectively). [(125)I]PYY binding to COS-7 cells expressing either the Y5(L) or the Y5(S) 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 Y5(L) and Y5(S) mRNAs, as determined by reverse transcription-PCR analysis, indicated that expression of Y5(L) mRNA occurs in a tissue-specific manner. Finally, we have shown that the two AUG triplets contained in the 5' untranslated region of Y5(L) mRNA did not affect receptor expression.