title: A novel non-opioid binding site for endomorphin-1.
abstract: Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the μ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the μ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the μ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd= 0.4 nM / Bmax= 120 fmol/mg protein) and lower (Kd= 8.2 nM / Bmax= 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no μ-opioid receptors, such as rodent cerebellum, or brain of μ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.|present address: UCL Institute of Ophthalmology, University College London, London, United Kingdom.
authors: I
authors: I
authors: Lengyel
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
authors: F
authors: F
authors: Toth
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.|present address: Feinberg Cardiovascular Research Institute, Northwestern University, Chicago, USA.
authors: D
authors: D
authors: Biyashev
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.|present address: Semmelweis University, First Department of Paediatrics, Laboratory of Metabolic Screening, Budapest, Hungary.
authors: I
authors: I
authors: Szatmari
authors: Unite de Regulation des Genes et Signalisation Cellulaire, INSERM U-99, Hopital Henri Mondor, Creteil, France.|present address: Department of Physiological Chemistry, University Medical Centre of the Johannes Gutenberg University, Mainz, Germany.
authors: K
authors: K
authors: Monory
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
authors: C
authors: C
authors: Tomboly
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
authors: G
authors: G
authors: Toth
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary. benyhe@brc.hu.
authors: S
authors: S
authors: Benyhe
authors: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
authors: A
authors: A
authors: Borsodi
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008297:Male; D018345:Mice, Knockout; D009292:Narcotic Antagonists / Q000494:pharmacology; D009479:Neuropeptides / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D011869:Radioligand Assay; D017208:Rats, Wistar; D017450:Receptors, Opioid, mu / Q000235:genetics
chemical_list: D000701:Analgesics, Opioid; D009292:Narcotic Antagonists; D009479:Neuropeptides; D009842:Oligopeptides; C404943:Oprm protein, mouse; D017450:Receptors, Opioid, mu; C105293:endomorphin 1; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases