Our understanding of the 5-ht_5A receptor, in comparison to other members of the 5-hydroxytryptamine (5-HT) receptor family, is limited. Predicted to be a G-protein coupled receptor, previous studies suggest it is expressed exclusively in the CNS (Rees et al., 1994), although with no clear coupling to a G-protein signalling pathway in native tissue, the 5-ht_5A receptor is merely referred to as a gene product (Barnes & Sharp, 1999). To investigate this protein further, we have cloned the 5-ht_5A receptor sequence and tagged the expressed protein with green fluorescent protein (GFP).

The 5-ht_5A receptor sequence was cloned from human lymphocyte genomic DNA into the expression vector pEGFP-N1. Subsequent removal of a stop codon allowed expression of the 5-ht_5A receptor with GFP coupled to the C-terminus (5-ht_5A-GFP). Transient expression of either protein was achieved in COS-7 cells using PolyFect (Qiagen) as the transfection reagent. Standard membrane preparations from transfected and non-transfected cells were subject to standard radioligand binding studies using [³H]5-HT (buffer; 50 mM Tris, 10 mM MgCl₂, 1 mM EGTA, pH 7.4).

Whilst previous studies have used exogenous compounds to label the 5-ht_5A receptor (Barnes & Sharp, 1999), our studies demonstrate that [³H]5-HT is also a suitable radioligand. Thus, [³H]5-HT (12 concentrations between 0.4-100 nM; non-specific binding defined by the presence of LSD (10 µM)) labelled a saturable population of binding sites in membranes prepared from COS-7 cells expressing the 5-ht_5A protein. Specific binding was not apparent in membranes prepared form COS-7 cells transiently transfected with vector only. Specific [³H]5-HT binding was better fitted to a two-site, rather than a one-site, binding model, with affinities (pKd; mean±SEM) of 8.51±0.18 (high affinity site) and 7.39±0.04 (low affinity site). Typically, high affinity sites only represented approximately 10 % of the specific labelled sites.

The pharmacological profile of the high affinity [³H]5-HT binding sites assessed by competition binding (3-5 nM [³H]5-HT; rank order of potency (pKi; mean±SEM, n=3) 5-carboxamidotryptamine (10.23±0.20) > LSD (8.73±0.19) > 5-methoxytryptamine (8.19±0.12) > clozapine (7.35±0.14) > dihydroergotamine (7.19±0.10) > mesulergine (6.42±0.20) >> fenfluramine (<6) losartan (<6) citalopram (<6) ondansetron (<6)) was comparable to previous reports concerning the pharmacology of the 5-ht_5A receptor using agonist radioligands (e.g. Rees et al., 1994).

5-ht_5A-GFP expression in COS-7 cells was distributed differentially and was absent from the nucleus. Interestingly, high affinity agonist binding (5-HT, 5-CT) was comparable in membranes prepared from COS-7 cells expressing the 5-ht_5A-GFP construct, suggesting that the coupling of GFP to the C-terminus of h5-5-ht_5A may be useful to investigate subcellular localisation of the agonist accepting 5-ht_5A receptor.

Barnes & Sharp, Neuropharmacology, 38, 1083-1152, 1999.
Rees, et al., FEBS Letters, 355, 242-246, 1994.