Cloning of two melanocortin (MC) receptors in spiny dogfish

Autor: Helgi B. Schiöth, Maja Löwgren, Susanne Stier, Aneta Ringholm, Davids Fridmanis, Maija Slaidina, Janis Klovins, Tatjana Haitina
Rok vydání: 2004
Předmět:
medicine.medical_specialty
Green Fluorescent Proteins
Molecular Sequence Data
CHO Cells
Adrenocorticotropic hormone
Biology
Polymerase Chain Reaction
Biochemistry
Cell Line
Radioligand Assay
gamma-MSH
Adrenocorticotropic Hormone
Cricetinae
Internal medicine
Cyclic AMP
Escherichia coli
medicine
Animals
Humans
Potency
Bacteriophages
Tissue Distribution
Amino Acid Sequence
Melanocyte-Stimulating Hormones
Cloning
Molecular
Receptor
Phylogeny
Gene Library
chemistry.chemical_classification
Spiny dogfish
Dose-Response Relationship
Drug
Sequence Homology
Amino Acid
Reverse Transcriptase Polymerase Chain Reaction
Chinese hamster ovary cell
HEK 293 cells
Sequence Analysis
DNA
biology.organism_classification
Molecular biology
Introns
Amino acid
Blotting
Southern
Kinetics
Endocrinology
chemistry
Dogfish
Receptor
Melanocortin
Type 4
Melanocortin
Peptides
Receptor
Melanocortin
Type 3
Zdroj: European Journal of Biochemistry. 271:4320-4331
ISSN: 1432-1033
0014-2956
DOI: 10.1111/j.1432-1033.2004.04374.x
Popis: We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.
Databáze: OpenAIRE
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