Isoldi MC et al. (2010), Light modulates the melanophore response to alp...

XB-ART-39971

Gen Comp Endocrinol 2010 Jan 01;1651:104-10. doi: 10.1016/j.ygcen.2009.06.014.

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Light modulates the melanophore response to alpha-MSH in Xenopus laevis: an analysis of the signal transduction crosstalk mechanisms involved.

Isoldi MC , Provencio I , Castrucci AM .

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Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X. laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane.

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Species referenced: Xenopus laevis
Genes referenced: akap12 akap13 camp pomc ppp3ca
GO keywords: melanocyte-stimulating hormone activity [+]

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	Fig. 1. Xenopus melanophores were seeded in 96-well plates and kept in the dark for a minimal of 3 days. cAMP was determined in Xenopus melanophores in the dark and after 1, 5, and 10 min light exposure, in the absence or presence of 10−8 M α-MSH. Each bar is the mean (n = 6), ±SE, fmol/104 cells. (a) Significantly different from the respective control in the absence of the hormone. (b) Significantly different from 10−8 M α-MSH in the dark. p < 0.01.
	Fig. 2. Partial amino acid sequences of Xenopuslaevis and human adenylyl cyclase IX (AC9). Identical residues in bold constitute the catalytic domains I and II (CLUSTALW 1.83).
	Fig. 3. Calcineurin activity in Xenopus melanophores in the dark, and after 1 and 5 min light exposure. Each point is the mean (n = 6), ±SE, phosphate nmol/106 cells, at the times noted.
	Fig. 4. Light or 10−8 M α-MSH-evoked pigment granule dispersion in Xenopus melanophores in the absence or presence of 5 × 10−6 M St-Ht31, the disrupting agent of AKAP–PKA complex. Each bar is the mean (n = 6), ±SE, absorbance (1000×) of light- or MSH-stimulated cells. ∗Significantly different (p < 0.001) from MSH-evoked response in the absence of St-Ht31.
	Fig. 5. Western blot of PKA catalytic subunit from Xenopus melanophores, previously co-immunoprecipitated with gravin. (A) Dark control, (B) 5 min 10−7 M α-MSH, and (C) 5 min of light exposure.