Neuropepfides (1997) 31 (6), 565-571 © HarcourtBrace and CompanyLtd 1997 Characterization of the binding of MSH.B, HP.228, GHRP.6 and 153N-6 to the human melanocortin receptor subtypes H. B. SchiSth*, R. Muceniece*, J. E. S. Wikberg* *Department of Pharmaceutical Pharmacology, Uppsala University, Uppsala, Sweden *Laboratory of Pharmacology, Institute of Organic Synthesis, Riga, Latvia Summary We determined the binding affinities of the MSH analogues MSH-B, HP-228 and 153N-6 and of the enkephalin analogue GHRP-6 on a single eukaryotic cell line transiently expressing the human MC1, MC3, MC4 and MC5 receptors. Moreover, we tested the binding and cAMP response of MSH-B in comparison with (z-MSH on murine B16 melanoma cells. Our results indicate that MSH-B has a potency similar to that of c~-MSH and that these two peptides induce similar cAMP responses in murine B16 melanoma cells. HP-228 has its highest affinity for the MC1 receptor. For the other receptors, it has slightly higher affinity for the MC5 receptor than for the MC3 and MC4 receptors. 153N-6 was found to be selective for the MC1 receptor. GHRP-6 was found to bind to the MC1 and the MC5 receptors despite its low structural homology with o~-MSH. [D-Lysa]GHRP-6 bound to all the four MC receptors with similar affinities. The structurally related Met-enkephalin and the functionally related GHRH, as well as LHRH and somatostatin-14 did not bind to these MC receptors. The low affinity of the GH-releasing/enkephalin peptides may indicate that they do not interact with the MC receptors at pharmacologically relevant concentrations. INTRODUCTION The melanocortin peptides are known primarily for their role in skin pigmentation and regulation of steroid production in the adrenal gland. In addition, the melanocortins, which include the natural ~z-, [~- and 7- MSH and ACTH, have a broad array of other physiologi- cal functions which are much less understood. These include effects on behaviour, memory, thermoregulation, pain perception, nerve regeneration, inflammation, blood pressure and parturition5 ,2 Cloning of five melanocortin (MC1-5) receptor sub- types has opened new possibilities to elucidate the physiological actions of the melanocortins and their receptors. >7 The MC1 receptor has high affinity for a-MSH, s plays an important role for pigmentation and is Received 25 June 1997 Accepted 18 August 1997 Correspondence to: Helgi B. Schi6th and Jarl E. S. Wikberg, Department of Pharmaceutical Pharmacology, Biomedical Center, Box 591,751 24 Uppsala, Sweden. Fax: + 46 18 559718; E-mail: helgis@bmc.uu.se, jarl. wikberg @farmbio.uu.se expressed in melanoma cells.3,4 The MC2 receptor is the ACTH receptor; it binds ACTH with high affinity but not the MSH peptides9 and is expressed in the adrenal gland.4 The physiological roles of the other three MC receptors, whose existence was not known prior to their cloning, have been much less characterized. The MC3 receptor is expressed mainly in the brain but has also been detected in peripheral tissues like the gut, placenta and heart, s,~°,1~ The MC4 receptor is found only in the brain and has recently been related to weight homeostasis. 12,~3 The MC5 receptor is found in the brain and has also a wide peripheral distribution but still has a much less charac- terized physiological role. ~4,15 a-MSH is selective for the MC1 receptor and ACTH is selective for the MC2 receptor, but none of the natural MSH peptides or other hormones are know to be selective for the newly discovered MC3, MC4 and MC5 recep- tors.8,9,~<~7 There are only a few reports on specific synthetic analogues for these subtypesY,~9More basic knowledge is needed about the binding of the MC receptor subtypes to MSH peptides to elucidate the subtype-specific properties which may allow construction of selective compounds. 565