Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor

Abstract

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Figure 1

Left panel: agarose gel electrophoresis of RT–PCR products of cDNA synthesized from porcine brain cortex (see Methods). M, Φ×174 DNA/HaeIII marker; Lanes 1, RT–PCR product of 625 bp using β-actin primers; Lane 2, RT–PCR product of approximately 1200 bp obtained using forward and reverse primers of 5-HT_1D receptors. A control without template cDNA (in absence of reverse transcriptase) did not show any signal (not included in the figure). Right panel: agarose gel electrophoresis of recombinant plasmid with 5-HT_1D receptor cDNA. M, Φ×174 DNA/HaeIII marker; Lane 1, recombinant plasmid DNA restricted with EcoRI enzyme and showing a DNA insert of approximately 1200 bp; Lane 2, non-digested recombinant plasmid DNA. The size (bp) of three marker bands is indicated in the left margin.

Figure 2

Nucleotide and deduced amino acid (in bold) sequences of the pig 5-HT_1D receptor (GenBank accession number: AF 117655). The computer predicted (software DNAMAN, version 3.2, Lynnon Biosoft^©) transmembrane domains I–VII (underlined) and putative N-glycosylation (★), protein kinase A phoshorylation (▪) and protein kinase C phosphorylation (•) sites are indicated in the sequence.

Figure 3

Sequence of inverse-PCR amplified products (in bold letters) showing a 100% homology at both 5′ and 3′ ends with the sequence of the cDNA identified from the pig brain cortex (in normal letters). The 5′ and 3′ terminal sequences are identified in boxes, while the inverse PCR primers are underlined.

Figure 4

Comparison of amino acid sequences of the pig 5-HT_1D receptor with human (GenBank accession number M89955), rabbit (Z50162), guinea-pig (X94436), mouse (X94908), rat (M89953) and dog (X14049) 5-HT_1D receptors (software DNAMAN, version 3.2, Lynnon Biosoft^©). The arrows drawn across the amino acid sequence indicate the seven transmembrane regions. Shaded boxes show identity across the different species.

Figure 5

Regression analysis of pK_i values (affinity constants) of 5-HT receptor ligands for the cloned pig 5-HT_1D receptor (see Table 1 for ligands and data) against the cloned human 5-HT_1D (upper panels) and 5-HT_1B (lower panels) receptors (data from Pauwels et al., 1996; Wurch et al., 1998), using either [³H]-5-CT (left panels) or [³H]-GR125743 (right panels). The Spearman correlation coefficient r_s, calculated by using SlideWrite plus for Windows (Advanced Graphics Software, Encinitas, CA, U.S.A.), is listed in each panel. The compounds clearly falling outside the regression line (BRL15572 and SB224289) are identified in the graphs.

Figure 6

Increase in [³⁵S]-GTPγS binding, as percentage of the response to 10 μM 5-HT, by zolmitriptan in CHO-K₁ cells co-transfected with the cloned pig 5-HT_1D receptor and rat G_αilCys³⁵¹Ile protein. The basal [³⁵S]-GTPγS binding (277±37 fmol mg⁻¹ protein) was increased by 5-HT (10 μM) to 399±77 fmol mg⁻¹ protein (44%) and decreased by ketanserin (10 μM) to 220±23 fmol mg⁻¹ protein (−25%).