PROJECT ABSTRACT Copper is an essential micronutrient in biology, playing fundamental roles in processes such as respiration and antioxidant defense. The context in which a copper ion resides is vital to its activity, and its miscompartmentalization and imbalances is associated with a host of disorders. While technologies have emerged to investigate intracellular copper dynamics, the extracellular copper pool is relatively under- characterized. The parent grant of this supplement seeks to elucidate the speciation of metals in the extracellular space, with a focus on the interplay of metals with another dynamic component in the extracellular environment, hormones. The main research of this objective focuses on a particular peptide with hormone-like functions, C- peptide, a 31-mer derived from the proinsulin protein. Historically undervalued as an innocent secondary marker for insulin, C-peptide is of increasing interest for its bioactivity independent of insulin. However, its advancement has been hampred by lack of mechanistic understanding. Work under the parent project has shown that C- peptide can directly bind to copper, an event that can modulate C-peptide's activity. In Aim 1 of this proposal, we seek to determine whether and how C-peptide and its truncates affect copper trafficking. Preliminary work suggests that the metal-binding interaction can shift the homeostatic balance of the metal. A combination of in vitro and in vivo work will seek to analyze the pathways that drive the interplay between C-peptide and copper. Aim 2 seeks to leverage the interactions of copper and C-peptide to identify a plasma membrane receptor for the peptide. While an orphan GPCR, GPR146, has been proposed as a partner for C-peptide, this claim remains debated. We hypothesize that metal interactions with the peptide, which is rich in negatively-charged residues, may be influential in receptor recognition. This aim will screen for C-peptide interactions with a library of orphan GPCRs in the presence and absence of transition metals to determine whether metals are integral to receptor recognition, and if so, identify a cognate receptor for the peptide. To achieve these research goals, the candidate to be supported by this supplement will be trained across disciplines with a multifaceted mentoring team to bring molecular insight to metalloendocrinological processes.