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

Orlow, Seth J.; Hotchkiss, Sara C.; Pawelek, John M.

doi:10.1002/jcp.1041420116

Cited by 36 publications

(25 citation statements)

References 23 publications

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“…Furthermore, we find that MMS and pTpT treatments increase the binding of '25I-a-MSH to the cell surface MSH receptor, as has been reported after UV irradiation (38,70). In the case of UV irradiation, a redistribution of MSH binding sites from an internal to external cellular localization was proposed (43). Although a change in receptor affinity for MSH could not be excluded, up-regulation of MSH binding on S91 cells by other agents, such as retinoic acid, appears to involve a change in the number of binding sites with no change in affinity for ligand (70).…”

Section: Discussionsupporting

confidence: 79%

DNA damage enhances melanogenesis.

Eller

Ostrom

Gilchrest

1996

Proc. Natl. Acad. Sci. U.S.A.

261

193

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Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin The prokaryotic response to UV irradiation, the so-called SOS response, is well documented and is now known to include the induction of a set of >20 genes involved in DNA repair and cell survival (reviewed in ref. 1). In this case, the single-stranded DNA generated after UV irradiation interacts with and activates a protease, the Rec A protein (2). Activated Rec A protein then cleaves and inactivates the repressors of specific genes, leading to their induction (2).In eukaryotic cells, the existence of a UV-induced DNA damage-responsive SOS-like system mediated by one common transcription regulator has been the subject of considerable controversy. Although a variety of genes are known to be induced by DNA damage (3-6), many of these genes are also induced by agents such as phorbol esters (3, 7) and by metabolic or oxidative stress (8-10). Because UV irradiation is reported, like phorbol esters, to activate protein kinase C directly (11,12) and to produce oxidative damage through generation of free radicals from membrane lipids and otherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. extranuclear cellular constituents, it cannot be determined whether the effects of UV are due directly to DNA damage or instead to other impacts on the cell. Indeed, two of the major UV-induced transcription factors, AP-1 and NF-KB, are now thought to initiate their responses at or near the plasma membrane (13,14).Perhaps the best-characterized example of DNA damagespecific gene induction involves a DNA repair enzyme, photolyase, encoded by the PHR1 gene in Saccharomyces cerevisiae (6). This gene is induced by a variety of DNA-damaging agents including UV light, methyl methanesulfonate (MMS) and 4-nitroquinoline 1-oxide (4-NQO). Up-regulation of PHR1 gene transcription is, at least in part, accomplished by the removal of a damage-responsive repressor which binds to a specific site in the 5' region of the gene (15).Another well-studied UV and DNA damage-inducible gene is the mammalian GADD45 gene. This gene is transcriptionally activated not only by UV irradiation but also by ionizing radiation and chemical agents that specifically cause base damage (10, 16). The induction of GADD45 by ionizing radiation is mediated by the p53 tumor suppressor protein and the ataxia telangiectasia gene product (17), but the UV-and base-damage responses are less well understood. ...

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Section: Discussionsupporting

confidence: 79%

DNA damage enhances melanogenesis.

Eller

Ostrom

Gilchrest

1996

Proc. Natl. Acad. Sci. U.S.A.

261

193

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“…Accordingly, we examined the effects of UVB on the outer surfaces of intact cells and on the internal binding sites described previously (Orlow et al, 1990). We did not examine the purified vesicular fractions, because the number of manipulations for purification of these vesicles made that experiment technically impractical.…”

Section: Resultsmentioning

confidence: 99%

“…We have shown that Cloudman melanoma cells exhibit internal binding sites for MSH and that expression of these sites is an important criterion for cellular responsiveness to MSH (Orlow et al, 1990). Here, with the use of cross-linking agents, as well as by immunologic means, we demonstrate that:…”

mentioning

confidence: 91%

Structural/functional relationships between internal and external MSH receptors: Modulation of expression in Cloudman melanoma cells by UVB radiation

Chakraborty

Orlow

Bolognia

et al. 1991

Journal Cellular Physiology

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Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells (Orlow et al: J. Cell. Physiol., 142:129-136, 1990). Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs (Bolognia et al: J. Invest. Derm., 92:651-656, 1989). Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.

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“…Because internalization of endogenous murine Mc1r likely targets the receptor for lysosomal degradation (44) and partially for delivery to the melanosomes (Ref. 40 and results presented in Fig. 3D), GRK6 would promote MC1R down-regulation and incorporation of internalized cargo to the melanosome.…”

Section: Discussionmentioning

confidence: 98%

“…2A, further supporting the use of HEK293 cells as a suitable heterologous model. Early work suggested that endogenous Mc1r expressed in mouse melanoma cells internalized to melanosomes after agonist binding (40). We extended these observations to human melanoma cells by using ␣PEP7h (29) for detection of the melanosomal enzyme tyrosinase (41), a suitable melanosomal marker.…”

Section: Grk6 Promotes Mc1r Internalization-in Hek293 Cells Transfectmentioning

confidence: 92%

Regulation of Human Melanocortin 1 Receptor Signaling and Trafficking by Thr-308 and Ser-316 and Its Alteration in Variant Alleles Associated with Red Hair and Skin Cancer

Sánchez-Laorden¹,

Jiménez‐Cervantes

García‐Borrón

2007

Journal of Biological Chemistry

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The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/ S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations. The human melanocortin 1 receptor (MC1R)3 is a G proteincoupled receptor preferentially expressed in epidermal melanocytes (1). Within melanocytes, MC1R regulates the amount and type of pigment production and is a major determinant of skin phototype, sensitivity to ultraviolet (UV) radiation-induced damage, and skin cancer risk (2). Interaction with its ligands, ␣ melanocyte-stimulating hormone (␣MSH) or adrenocorticotropin, stimulates cAMP synthesis via the G s protein. cAMP is responsible for most if not all the melanogenic actions of ␣MSH (3). These include activation of tyrosinase, the rate-limiting enzyme in melanin biosynthesis, and a switch from the production of light-colored and poorly photoprotective pheomelanins to synthesis of darker and more photoprotective eumelanins (4). Tyrosinase activation occurs mainly at the transcriptional level and is mediated by induction of microphthalmia, a helix-loophelix transcription factor (5). Melanin synthesis is confined in specialized organelles called melanosomes. The super-potent MC1R agonist norleucine 4 D-phenylalanine-7-melanocyte-stimulating hormone (NDP-MSH) also increases the number and size of melanosomes in mouse melanoma cells (6), s...

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

Cited by 36 publications

References 23 publications

DNA damage enhances melanogenesis.

DNA damage enhances melanogenesis.

Structural/functional relationships between internal and external MSH receptors: Modulation of expression in Cloudman melanoma cells by UVB radiation

Regulation of Human Melanocortin 1 Receptor Signaling and Trafficking by Thr-308 and Ser-316 and Its Alteration in Variant Alleles Associated with Red Hair and Skin Cancer

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