Improving women’s healthStructure and function of wild-type and mutant peptide models of the alpha2A adrenergic receptor.

Abstract

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and play a central role in cellular signaling. The molecular details of how these 7 transmembrane (TM)-helix containing proteins activate guanine nucleotide binding proteins (G proteins) are not known. However, mutagenesis studies show that residues in the intracellular loops (ILs) play an important role in G protein activation. Peptides from the ILs inhibit GPCR-G protein interactions and some of them mimic the receptor by activating G proteins. Since it is difficult to purify sufficient quantities of GPCRs for biophysical studies, I examined peptide models of the IL, including solving the high-resolution NMR structure of IL-2. First, we characterized fusion protein systems that allowed bacterial expression and small-scale purification of a 41-residue TM-spanning peptide from the alpha2A adrenergic receptor (alpha2AR), with the aim of producing isotope-labeled samples for NMR study. This work outlines the basis for future large-scale preparations. Next, we focused on identifying the roles of key residues in the alpha2AR. Three mutations in the receptor have different effects: D130I increases agonist-binding affinity for the alpha2AR ∼5-fold; R131Q severely impairs G protein activation; and T373K constitutively activates the alpha2AR. I made the corresponding mutations in two functionally active 32-residue and 19-residue peptides derived from IL-2 and IL-3, named T6-I3C and T3-12. The R131Q and T373K mutations have minimal structural and functional effects in the peptides, arguing that these residues in the alpha2AR play a structural role forming intramolecular contacts. However, the D1301 mutation induces changes in secondary structure and enhances G protein activation by T3-12 suggesting that this mutation may cause direct, local changes in the receptor. Using circular dichroism spectroscopy, we showed that T3-12 readily forms helical structure, whereas T6-I3C adopts a more disordered conformation. NMR studies on T3-12 in micelles showed a 5-residue TM helix and a 15-residue cytosolic helix that were separated by a linker whose structure changed to helix with the D1301 mutation. The linker and extensive cytosolic helix are novel findings that are not present in current GPCR models and should lead to changes in our understanding of GPCR structure and function.