Wild-type human prolactin (hPRL) is a 23 kDa peptide hormone which has many important biological functions mostly in the reproductive system. It functions by binding to the cell surface human prolactin receptors which are located on many tissues. As the hormone binds to its receptor, heterodimeric and heterotrimeric complexes will form in a sequential manner. Their binding reaction will then trigger the JAK-STAT signaling cascade.
hPRL is a four-helical bundle protein. Previous work in our laboratory has shown that deletion of mini-helix 1, creating a protein called Δ41-52 hPRL, by site-directed mutagenesis has reduced the lactogenic activity. Mammary cells and human breast cancer cells contain human prolactin receptors and can secrete prolactin in an autocrine and paracrine manner that may contribute to the growth of breast cancer cells.
Therefore, our laboratory wishes to develop a strong human prolactin antagonist and Δ41-52 hPRL is a candidate compound. This work shows for the first time that Δ41-52 hPRL is a potent human prolactin antagonist in two biological assays, namely, the FDC-P1 lactogenic assay using Alamar blue, a vital dye, to estimate cell number and by measuring apoptosis through flow cytometry.
This dissertation also presents original work performed to compare the physical properties between wild-type hPRL and Δ41-52 hPRL with denaturation studies followed by several types of spectroscopy. First, absorbance spectroscopy was used to obtain information on the aromatic acids, disulfide bonds and protein aggregation. Second, fluorescent spectroscopy was used to compare the packing of the hydrophobic core using tryptophan as an indicator. Lastly, circular dichroism was used to study the secondary structure of the protein which is mostly helical structure.
In thermal denaturation studies, Δ41-52 hPRL is more stable than wild-type hPRL in every aspect except in the secondary structure where Δ41-52 hPRL gradually loses its helical content. In a pH denaturation study, the helical structure of Δ41-52 hPRL shows greater stability than wild-type hPRL. A study performed with fluorescent spectroscopy on the extracellular domain of the human prolactin receptor shows that it has a very low melting temperature of only 45°C.
This dissertation also presents findings on the binding reaction of Δ41-52 hPRL with the hPRL receptor by using fluorescent spectroscopy. The forward binding reaction reaches equilibrium in approximately 1 hour. The dissociation reaction occurred too rapidly to be recorded by our equipment.