A profile of the residues in the first intracellular loop critical for Gs-mediated signaling of human prostacyclin receptor characterized by an integrative approach of NMR-experiment and mutagenesis.
The first intracellular loop (iLP1, residues 39-51) of human prostacyclin receptor (IP) was proposed to be involved in signaling via its interaction with the Galphas protein. First, evidence of the IP iLP1 interaction with the C-terminus of the Galphas protein was observed by the fluorescence and NMR spectroscopy using the synthetic peptide (Galphas-Ct) mimicking the C-terminal 11 residues of the Galphas protein in the presence of a constrained synthetic peptide mimicking the IP iLP1. Then, the residues (Arg42, Ala44, and Arg45) in the IP iLP1 peptide possibly involved in contacting the Galphas-Ct peptide were initially assigned by observation of the significant proton resonance shifts of the side chains of the constrained IP iLP1 peptide using 2D (1)H NMR spectroscopy. The results of the NMR studies were used as a guide for further identification of the residues in the IP important to the receptor signaling using a recombinant protein approach. A profile of the residues in the IP iLP1, including the residues observed from the NMR studies involved in the Galphas mediated signaling, was mapped out by mutagenesis. According to our results, it can be predicted that the seven residues (Arg42-Ala48) with the conserved Arg45 at the center will form an epitope with a specific conformation involved in the Galphas mediated signaling. The conservation of the basic residues (Arg45 in the IP) in all of the prostanoid receptors suggests that the iLP1 regions of the other prostanoid receptors may also contain the epitopes important to their signaling.
Zhang
Lihai
L
Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, The University of Texas Health Science Center, Houston, Texas 77030, USA.
Huang
Gangxiong
G
Wu
Jiaxin
J
Ruan
Ke-He
KH
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000502:physiology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D044006:Receptors, Epoprostenol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D044006:Receptors, Epoprostenol; D011994:Recombinant Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs