Abstract
Melanocortin 2 receptor accessory protein (MRAP) facilitates trafficking of melanocortin 2 (MC2) receptors and is essential for ACTH binding and signaling. MRAP is a single transmembrane domain protein that forms antiparallel homodimers. These studies ask when MRAP first acquires this dual topology, whether MRAP architecture is static or stable, and whether the accessory protein undergoes rapid turnover. To answer these questions, we developed an approach that capitalizes on the specificity of bacterial biotin ligase, which adds biotin to lysine in a short acceptor peptide sequence; the distinct mobility of MRAP protomers of opposite orientations based on their N-linked glycosylation; and the ease of identifying biotin-labeled proteins. We inserted biotin ligase acceptor peptides at the N- or C-terminal ends of MRAP and expressed the modified proteins in mammalian cells together with either cytoplasmic or endoplasmic reticulum-targeted biotin ligase. MRAP assumed dual topology early in biosynthesis in both CHO and OS3 adrenal cells. Once established, MRAP orientation was stable. Despite its conformational stability, MRAP displayed a half-life of under 2 h in CHO cells. The amount of MRAP was increased by the proteasome inhibitor MG132 and MRAP underwent ubiquitylation on lysine and other amino acids. Nonetheless, when protein synthesis was blocked with cycloheximide, MRAP was rapidly degraded even when MG132 was included and all lysines were replaced by arginines, implicating non-proteasomal degradation pathways. The results show that although MRAP does not change orientations during trafficking, its synthesis and degradation are dynamically regulated.
Highlights
The hypothalamic/pituitary/adrenal axis comprises a classical endocrine loop in which hypothalamic corticotropin-releasing hormone stimulates synthesis and release of ACTH from corticotropes of the anterior pituitary gland, ACTH stimulates glucocorticoid production in the adrenal gland, and glucocorticoids exert feedback control centrally
We developed an approach that capitalizes on the specificity of bacterial biotin ligase, which adds biotin to lysine in a short acceptor peptide sequence; the distinct mobility of Melanocortin receptor accessory protein (MRAP) protomers of opposite orientations based on their N-linked glycosylation; and the ease of identifying biotin-labeled proteins
The experiments described here do not explain what advantages are conferred by the conserved MRAP structure but they do establish that MRAP topology is stable from the time of MRAP biosynthesis to the time of MRAP degradation
Summary
The hypothalamic/pituitary/adrenal axis comprises a classical endocrine loop in which hypothalamic corticotropin-releasing hormone stimulates synthesis and release of ACTH from corticotropes of the anterior pituitary gland, ACTH stimulates glucocorticoid production in the adrenal gland, and glucocorticoids exert feedback control centrally. The melanocortin 2 (MC2) (ACTH) receptor is one of five structurally related G protein-coupled melanocortin receptors, MC1 through MC5, all of which are activated by peptides derived from pro-opiomelanocortin, or POMC. The MC2 receptor was found to differ from the other four melanocortin receptors in two important ways It could not be activated by α-, β-, or γ-MSH but only by the longer ACTH peptide. It was not functional unless it was expressed in adrenal or melanoma cells. This led to speculation that the MC2 receptor required an additional protein present in a limited number of cell types
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