Internal binding sites for MSH: Analyses in wild-type and variant Cloudman melanoma cells

Journal Article · Mon Jan 01 04:00:00 EST 1990 · Journal of Cellular Physiology; (USA)

DOI:https://doi.org/10.1002/jcp.1041420116· OSTI ID:7192051

Orlow, S J; Hotchkiss, S; Pawelek, J M ^[1]

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1. Yale Univ. School of Medicine, New Haven, CT (USA)

Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient and centrifuged; fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.

• OSTI ID: 7192051
• Journal Information: Journal of Cellular Physiology; (USA), Journal Name: Journal of Cellular Physiology; (USA) Vol. 142:1; ISSN 0021-9541; ISSN JCLLA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CENTRIFUGATION
DAYS LIVING RADIOISOTOPES
DISEASES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME ACTIVITY
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MELANOMAS
MEMBRANE PROTEINS
MICE
NEOPLASMS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PHENOTYPE
PITUITARY HORMONES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
TUMOR CELLS
ULTRACENTRIFUGATION
VERTEBRATES