Interaction of neurochondrin with the melanin-concentrating hormone receptor 1 interferes with G protein-coupled signal transduction but not agonist-mediated internalization.

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.

MYND domain specific interaction of the melanin-concentrating hormone receptor 1 interacting zinc-finger protein with alpha- and beta-tubulin.

MIZIP was originally identified as a highly conserved zinc-finger protein from human brain interacting with the C-terminus of the melanin-concentrating hormone receptor 1. However, the cellular functions of MIZIP are still not known. Here, we focussed on the identification of associated proteins using affinity purification from human cells. This resulted in the identification of alpha- and beta-tubulin. The interaction was confirmed in vitro and in vivo using GST pull-down and immunoprecipitation assays, and was mapped to the MYND zinc-finger of MIZIP and to the N-terminus of tubulin. Immunoprecipitation and immunocytochemistry analyses demonstrate that MIZIP binds to tubulin but not to cellular microtubules in vivo and that ectopic expression of MIZIP does not interfere with the overall structure of the microtubular cytoskeleton. Our results suggest that MIZIP might play an important role in mammalian cells by associating with tubulin and thus might provide a link between MCHR1 and tubulin functions.

Immunohistochemical distribution of MIZIP and its co-expression with the Melanin-concentrating hormone receptor 1 in the adult rodent brain.

We have recently identified a Melanin-concentrating hormone receptor 1 interacting zinc-finger protein (MIZIP) from a human brain cDNA library. Here, we report the generation of a specific antibody against MIZIP and its distribution in rodent tissues using immunoblotting and immunohistochemical techniques. MIZIP was detected as a 27 kDa protein in brain, liver, and skeletal muscle, and to a lower extend, in lung, testis, and heart. Subcellular fractionation of adult mouse brain revealed the presence of MIZIP and MCHR1 in the cytoplasmic, membrane, and synaptosomal fraction, but not in a postsynaptic density preparation. In cultured rat, embryonic hippocampal neurons MIZIP is somatodendritically localized. In the adult rodent brain, MIZIP is widely distributed. High levels of expression were detected in brain regions involved in olfaction, feeding behavior, sensorimotor integration, and learning and memory, for example, the olfactory bulb, the olfactory tubercle, the caudate putamen, the thalamus and hypothalamus, the nucleus accumbens, the cerebral cortex, the hippocampus formation, and the cerebellum. Co-expression of MIZIP and MCHR1 was observed, for example, in pyramidal neurons of the cerebral cortex and hippocampus, in neurons of the olivary nucleus, lateral hypothalamus, nucleus accumbens, caudate putamen, pontine, and mesencephalic trigeminal nucleus. However, there are also differences in the expression patterns, for example, high expression of MCHR1 was detected in the lateral habenula, but no expression of MIZIP. These data support the notion that MIZIP might interact with MCHR1 in a cell type specific manner in vivo, suggesting a role in the regulation of MCH signalling in distinct regions of the mammalian brain.