Abstract
Melanin concentrating hormone (MCH) and neuropeptide EI (NEI) are two peptides produced from the same precursor in mammals, by cleavage at the Arg145-Arg146 site and the Lys129-Arg130 site, respectively. We performed co-localization studies to reveal simultaneously the expression of MCH mRNA and proconvertases (PCs) such as PC1/3 or PC2. In the rat hypothalamus, PC2 was present in all MCH neurons, and PC1/3 was present in about 15-20% of these cells. PC1/3 or PC2 was not found in MCH-positive cells in the spleen. In GH4C1 cells co-infected with vaccinia virus (VV):pro-MCH along with VV:furin, PACE4, PC1/3, PC2, PC5/6A, PC5/6B, or PC7, we observed only efficient cleavage at the Arg145-Arg146 site to generate mature MCH. Co-expression of pro-MCH together with PC2 and 7B2 resulted in very weak processing to NEI. Comparison of pro-MCH processing patterns in PC1/3- or PC2-transfected PC12 cells showed that PC2 but not PC1/3 generated NEI. Finally, we analyzed the pattern of pro-MCH processing in PC2 null mice. In the brain of homozygotic mutants, the production of mature NEI was dramatically reduced. In contrast, MCH content was increased in the hypothalamus of PC2 null mice. In the spleen, a single large MCH-containing peptide was identified in both wild type and PC2 null mice. Together, our data suggest that pro-MCH is processed differently in the brain and in peripheral organs of mammals. PC2 is the key enzyme that produces NEI, whereas several PCs may cleave at the Arg145-Arg146 site to generate MCH in neuronal cell types.
Highlights
Melanin concentrating hormone (MCH) and neuropeptide EI (NEI) are two peptides produced from the same precursor in mammals, by cleavage at the Arg145Arg146 site and the Lys129-Arg130 site, respectively
Based on their tissue distribution and intracellular localization, the mammalian subtilisin/kexin-like serine proteinases can be subdivided into four classes: (i) furin [6] and the recently discovered PC7 [7] process precursors that reach the cell surface via the constitutive secretory pathway; (ii) PC1/3 and PC2 process precursors whose products are stored in neuroendocrine secretory granules; (iii) PACE4 [9], the isoforms PC5/6-A (10 –12), and PC5/6-B [13], which are expressed in both endocrine and nonendocrine cells, conceivably process precursors in both the constitutive and regulated secretory pathways; (iv) PC4, which is predominantly synthesized in testicular germ cells [14, 15], appears important for fertilization [16]
Co-localization of MCH mRNA and Proconvertases in the lateral hypothalamic area (LHA)—Using 33P-labeled RMCH2 oligoprobe on the one hand and different proconvertase antisera on the other, we simultaneously examined the expression of MCH mRNA and the MCH-IR Peptideb
Summary
Co-localization Studies: in Situ Hybridization/Immunohistochemistry—Frozen sections (12 m-thick) of brain, spleen, or testis from adult Wistar rats (180 g weight) were cut on a cryostat at Ϫ20 °C and thaw-mounted on silane-coated slides. Expression of MCH mRNA and pro-MCH-derived peptides (MCH, NEI) was determined by RT-PCR [47] and by RIA coupled to RP-HPLC as described below. Tissues were used either for RNA or peptide extraction as described below. Semiquantitative RT-PCR analysis was performed using competition between reverse-transcribed RNA from tissues and a short template (MCH⌬) [48] amplified with RMCH2 (5Ј-CCAACAgggTCggTAgACTCgTCCCAgCAT-3Ј) and RMCH60 (5Ј-gAgCggCTTCATgAAggATgAC-3Ј) primers. Rat brain sections were hybridized with 33P-labeled RMCH2 oligonucleotide, and PC1/3 or PC2 protein was detected using specific antisera as described under “Materials and Methods.”. Peptide Extraction—Virus-infected GH4C1 cells were sonicated in 1 ml of 0.06 N HCl, 0.1% (v/v) trifluoroacetic acid solution. The NEI antiserum recognized well the amidated NEI peptide and NEI-GR, but NEI-GRR cross-reacted Ͻ0.1% with this antiserum [43, 45]
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