Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes, and interaction of MC4R and melanocortin receptor accessory protein 2 (MRAP2) is suggested to play pivotal role in energy balance of vertebrates. Topmouth culter (Culter alburnus) is an economically important freshwater fish in China. Herein we cloned culter mc4r, mrap2a, and mrap2b. Culter mc4r consisted of a 981 bp open reading frame encoding a protein of 326 amino acids. qRT-PCR revealed that mc4r, mrap2a, and mrap2b were primarily expressed in the central nervous system. In the periphery, mc4r and mrap2b were expressed more widely in the male, while mrap2a was expressed more widely in the female. Culter MC4R could bind to four peptide agonists and increase intracellular cAMP production dose dependently. Culter MC4R was constitutively active in both cAMP and ERK1/2 pathways, which was differentially regulated by culter MRAP2a and MRAP2b. Culter MRAP2a significantly increased B max and decreased agonist-stimulated cAMP, while MRAP2b increased cell surface and total expression but did not affect B max and agonist-stimulated cAMP. These results will aid the investigation of the potential physiological processes that MC4R might be involved in topmouth culter.
Melanocortin-3 receptor (MC3R) is a regulator of energy homeostasis, and interaction of MC3R and melanocortin-2 receptor accessory protein 2 (MRAP2) plays a critical role in MC3R signaling of mammals. However, the physiological roles of MC3R in teleosts are not well understood. In this study, qRT-PCR was used to measure gene expression. Radioligand binding assay was used to study the binding properties of topmouth culter MC3R (caMC3R). Intracellular cAMP generation was determined by radioimmunoassay and caMC3R expression was quantified with flow cytometry. We showed that culter mc3r had higher expression in the central nervous system. All agonists could bind and stimulate caMC3R to increase dose-dependently intracellular cAMP accumulation. Compared to hMC3R, culter MC3R showed higher constitutive activity, higher efficacies and Rmax to α-MSH, des-α-MSH, and ACTH. Both caMRAP2a and caMRAP2b markedly decreased caMC3R basal cAMP production. However, only caMRAP2a significantly decreased cell surface expression, Bmax and Rmax of caMC3R. Expression analysis suggested that MRAP2a and MRAP2b might be more important in regulating MC3R/MC4R signaling during larval period, and reduced mc3r, mc4r, and pomc expression might be primarily involved in modulation of MC3R/MC4R in adults. These data indicated that the cloned caMC3R was a functional receptor. MRAP2a and MRAP2a had different effects on expression and signaling of caMC3R. In addition, expression analysis suggested that MRAP2s, receptors, and hormone might play different roles in regulating culter development and growth.
activin β A and β B from diploid and allotriploid crucian carp were cloned.The differential expression of activin β A and β B genes in female allotriploid and diploid red crucian carp Carassius auratus red var. were studied and found to be expressed in all the tested tissues; particularly, the expression of activin β A and β B was elevated in the ovaries of allotriploids and differential expression in pituitaries during the nonbreeding season and the breeding season period. The immunohistochemistry indicated that the abnormal triploid ovaries were dominated by small oogonium-like cells with dense signals and that the elevated expression of activin β A and β B in the ovaries of allotriploids may be related to allotriploid sterility.
K E Y W O R D Sactivin β A , activin β B , allotriploid, Carassius auratus red. var., differential expression, sterility Activin is a member of the transforming growth factor-β (TGF-β) family of growth and differentiation factors (Vale et al., 1988). Activin was first isolated from gonadal fluids (Mathews, 1994) and commonly consists of three types: activin A, activin B and activin AB. Homodimerisation of the β A subunit generates activin A and the β B subunit generates activin B. The β A and β B subunits can also heterodimerise and form activin AB. In addition to the β A and β B subunits, β C and β E have been identified in mammals (Fang et al., 1996;Hotten et al., 1995) and a β D subunit has been described in the Africa clawed frog Xenopus laevis (Oda et al., 1995), but the β A and β B subunits have been researched more frequently (Woodruff, 1998).
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