Characterization of [Phe(13), Tyr(19)]-MCH analog binding activity to the MCH receptor.
Melanin concentrating hormone (MCH), a hypothalamic neuropeptide, is an important regulator of energy homeostasis in mammals. Characterization of an MCH specific receptor has been hampered by the lack of a suitable radioligand. The [Phe(13), Tyr(19)]-MCH analog has been shown by different investigators to bind specifically to cell lines of epithelial or pigment cell origin. Recently, using functional assays, the MCH receptor has been characterized as a seven transmembrane G-coupled protein initially identified as SLC-1. In the present study, we used tyrosine iodinated [Phe(13), Tyr(19)]-MCH analog, which stimulates food intake in a manner similar to that of MCH, as well as native MCH to conduct binding studies. Specific binding could not be demonstrated in intact cells of several cell lines, including A431 and B16. Specific binding associated with membranes localized to the microsomal, not the plasma membrane, fraction. Message for SLC-1 was absent in these cell lines, as assessed by Northern blot analysis. We conclude that cells previously reported to express the MCH receptor do not express SLC-1 and that both iodinated MCH and the [Phe(13), Tyr(19)]-MCH have a large component of non-specific binding. These ligands may be useful for binding studies in transfected cells with high levels of SLC-1 expression. However they do not appear to be suitable for screening for the MCH receptor as most cells demonstrate significant low affinity non-specific binding.
Kokkotou
E
E
Research Division, Joslin Diabetes Center and Harvard Medical School, 1 Joslin Place, Boston, MA 02215, USA.
Mastaitis
J W
JW
Qu
D
D
Hoersch
D
D
Slieker
L
L
Bonter
K
K
Tritos
N A
NA
Maratos-Flier
E
E
D000818:Animals; D001692:Biological Transport; D002294:Carcinoma, Squamous Cell; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005247:Feeding Behavior / Q000187:drug effects*; D006801:Humans; D007028:Hypothalamic Hormones / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008543:Melanins / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D008861:Microsomes / Q000378:metabolism; D010907:Pituitary Hormones / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011979:Receptors, Pituitary Hormone / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D007028:Hypothalamic Hormones; D008543:Melanins; D010907:Pituitary Hormones; D011979:Receptors, Pituitary Hormone; C092274:melanin-concentrating hormone receptor; C402828:melanin-concentrating hormone, Phe(13),Tyr(19)-; D004815:Epidermal Growth Factor; C019463:melanin-concentrating hormone