Investigation of metal modulation of oxytocin structure receptor-mediated signaling

Abstract

Oxytocin is a 9-amino acid peptide hormone. Since its discovery in 1954, it has most commonly been studied in relation to its role in stimulating parturition and lactation. However, it is now known that oxytocin has a widely diverse set of functions throughout the body including neuromodulation, bone growth, and inflammation. Previous research has suggested that divalent metal ions may be required for oxytocin activity, but the exact metal species and specific pathways have yet to be fully elucidated. In this work, we focus on characterizing copper and zinc bound forms of oxytocin and related analogs through far-UV circular dichroism. We report that Cu(ii) and Zn(ii) bind uniquely to oxytocin and all analogs investigated. Furthermore, we investigate how these metal bound forms may affect downstream signaling of MAPK activation upon receptor binding. We find that both Cu(ii) and Zn(ii) bound oxytocin attenuates the activation of the MAPK pathway upon receptor binding relative to oxytocin alone. Interestingly, we observed that Zn(ii) bound forms of linear oxytocin facilitate increased MAPK signaling. This study lays the foundation for future work on elucidating the metal effects on oxytocin's diverse bioactivity.

Fig. 1. Chemical structure of oxytocin (oxOT), reduced oxytocin (rOT), reduced Y2F-oxytocin (rOTF), and carbetocin.

Fig. 2. Circular dichroism spectra of 40 μM oxOT, rOT, rOTF, and carbetocin (15 mM phosphate, pH 7.4).

Fig. 3. Circular dichroism spectra of 40 μM oxOT, rOT, rOTF, and carbetocin in the absence or presence of one equivalent of either Cu(ii) or Zn(ii) (15 mM phosphate, pH 7.4).

Fig. 4. Electronic absorption spectra of (A) 200 μM oxOT (15 mM phosphate, pH 7.4) in the absence (apo) and presence of 200 μM CrCl₃, MnCl₂, FeCl₃, CoCl₂, NiCl₂, CuCl₂, ZnCl₂ and (B) CuCl₂ titrated into 200 μM oxOT (15 mM MOPS, pH 7.4) from sub-(0.5 eq.) to super-stoichiometric (5.0 eq.) ratios. CrCl₃, MnCl₂, FeCl₃, CoCl₂, and NiCl₂ have negligible changes to the spectrum of oxOT, whereas CuCl₂ alters the spectrum of oxytocin by blue-shifting the tyrosine band from 275 to 270 nm and forming a d–d band centered at 515 nm.

Fig. 5. Difference in ellipticity of the peaks in the circular dichroism spectra of oxOT and its analogs in the presence of Cu(ii) (light gray) or Zn(ii) (dark gray), calculated as the difference in apo-peptide and metal-treated peptide (Δ Ellipticity = Ellipticity (metal-treated) – Ellipticity (apo-treated)). Values were calculated for the peaks at (A) ≤200 nm (195 nm, 190 nm, 200 nm, and 190 nm for oxOT, rOT, rOTF, and carbetocin, respectively) to assess changes in the tail or overall structure and (B) the 225–230 nm range (225 nm, 230 nm, 230 nm, and 228 nm for oxOT, rOT, rOTF, and carbetocin, respectively) to assess changes in the conformation of the aromatic ring of the Tyr, Phe, or O-Me Tyr side chains.

Fig. 6. Densitometry of western blots for total and phosphorylated MAPK reveal metal-dependent modulation of MAPK activation by oxytocin (n = 3 to 4). Statistical significance of peptide alone compared to metal bound peptide was assessed by calculating p-values using unpaired t-test, * p < 0.05.