Characterisation of signalling by recombinant human neuromedain U receptors   Brighton P.J, Szekeres P.G* Aiyar, N+ & Willars G.B. Department of Cell Physiology and Pharmacology, University of Leicester, University Road, LE1 9HN and GlaxoSmithKline *Harlow, UK & +King of Prussia, PN, USA.

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Neuromedin U (NmU) is a neuropeptide, conserved across species, with putative roles in the regulation of blood flow, smooth muscle contraction, cancer, stress, anxiety, and obesity. However, its precise patho-physiological functions are unclear. Recently, two G-protein coupled receptors for NmU have been cloned having approximately 50% sequence homology. These receptors (NmU-R1 and NmU-R2) have distinct tissue distributions suggesting they may have different biological roles. Here, we have examined and compared some of the signalling properties of the recombinant human receptors.

The cDNAs encoding hNmU-R1 and hNmU-R2 were cloned separately into pCDN, and transfected into HEK-293 cells using standard methodology. Cell lines were selected and receptor expression determined by binding of human [¹²⁵I]-NmU-25 to membranes. Cell lines expressing hNmU-R1 (B_max 1.38±0.12 pmol mg^-1 protein, K_d 75±10 pM, n=3) and hNmU-R2 (B_max 1.96±0.24 pmol mg^-1 protein, K_d 176±18 pM, n=3) were selected for study. All data presented are mean±s.e.mean

Single cell imaging of intracellular [Ca²⁺] ([Ca²⁺]_i) in fluo-3 loaded cells expressing either receptor revealed robust (2-3 fold over basal), rapid (5s) peaks followed by lower (1.2-1.4 fold over basal) sustained phases in response to 10nM human NmU-25. Removal of extracellular Ca²⁺ abolished the sustained response while thapsigargin pre-treatment (1µM, 10min) abolished all responses. Determination of Ins(1,4,5)P₃ in cell populations by radioreceptor assay or in single cells by imaging of the eGFP-taggedPH domain of PLC-1 as a biosensor (Nash et al., 2001) also revealed similar peak and plateau responses. The accumulation of [³H]-labelled inositol phosphates against a Li⁺-block demonstrated an initial rapid (300-350% over basal min^-1) accumulation that became (at 20s) slower (50-60% over basal min^-1) but sustained for up to 1h. Accumulation was insensitive to pertussis toxin (100 ng ml^-1, 20h) and had pEC₅₀ values of 9.14±0.07 and 8.97±0.18 for hNmU-R1 and hNmU-R2 respectively (n=3). Immunoprecipitation of specific G-protein -subunits from cell membranes incubated with [³⁵S]-GTPµS (Akam et al., 1998) demonstrated that 10nM NmU caused a 4-fold increase in [³⁵S]-GTPµS binding to both G_q/11 and G_i but did not enhance binding to G_s (n=4).

Furthermore, in intact cells expressing either hNmU-R1 or hNmU-R2, NmU-25 inhibited forskolin (1µM)-stimulated accumulation of cAMP with pEC₅₀ values of 10.10±0.16 and 10.06±0.17 respectively (n=3). Activation of either receptor in the presence or absence of the phosphodiesterase inhibitor, IBMX, (500µM) did not elevate cAMP. Activation of either receptor resulted in the activation of ERK1/2 as determined by Western blotting with a phosphoERK-specific antibody. Maximal phosphorylation occurred after 5-10min and declined over the following 60min (n=4).

In summary, both hNmU-R1 and hNmU-R2 couple to both G_q/11and G_i in HEK-293 cells, and despite structural differences these receptors have similar signalling capability.

Akam E.C. et al. (1998) Brit. J. Pharmacol. 132, 950-958.
Nash. M.S. et al. (2001) Biochem. J. 356, 137-142.