| The protective actions of prostacyclin (PGI2) on the cardiovascular system, namely the promotion of vascular smooth muscle relaxation, along with inhibition of platelet aggregation, are mediated through a seven transmembrane-spanning G-protein coupled receptor (GPCR), known as the hIP or human prostacyclin receptor. This thesis sets forth to: (1) understand the molecular mechanisms by which the hIP interacts with ligand and transmits agonist-induced signal through the receptor protein, and (2) identify and characterize functional receptor variants, which may serve as predisposing and/or modifying factors of cardiovascular disease. (1) Using site-directed mutagenesis, this thesis identifies differential roles for particular transmembrane (TM) proline residues. In addition to providing critical kinks within TM alpha-helices, these amino acids may serve as molecular hinges or swivels, facilitating intra-alpha-helical movement required during receptor activation. (2) Using site-directed mutagenesis, thiol-reducing agents, and functional binding and activation assays, this thesis identifies a potential secondary, non-conserved disulfide bond in the extracellular domain, which may be important for receptor folding, trafficking and expression. The role of other conserved and non-conserved cysteine residues, located within the transmembrane (TM) domain, is also examined. (3) Using similar mutagenesis, along with functional binding assays, prostanoid receptor and ligand comparisons, and a three-dimensional, computer-generated homology model based upon the 2.8A crystal structure of rhodopsin, this thesis elucidates the unique ligand-binding pocket for the hIP. Four crucial points of receptor-ligand interaction were identified (from thirty initial mutations), including residues R279 (TMVII), F278 (TMVII), F95 (TMIII), and Y75 (TMII), which are predicted to interact with the C1-COOH group, central oxolane ring, o-tail, and C11-OH group of prostacyclin, respectively. (4) A related study determined the molecular requirements for receptor activation, within the transmembrane region of the hIP. Four distinct activation clusters were identified: (a) immediate binding-pocket cluster occupying TMII, TMIII, and TMVII, (b) P17-associated, proximal binding-pocket cluster residing predominately in TMII, (c) D60-associated, cytoplasmic-oriented cluster involving residues in TMI, TMII and TMVII, and (d) distal, aromatic cluster of TMIV and TMV. (5) Functional characterization of the first hIP polymorphisms identified in the SNP database (V25M and R212H), reveals a significant decrease in agonist-induced receptor activation at both pH 7.4 and 6.8 for the R212H variant. A defect in binding affinity was also observed for the R212H under acidotic conditions (binding affinity was also observed for the R212H under acidotic conditions (<pH 6.8). (6) Using genetic screening of both a general (four ethnic subgroups) and diseased (coronary artery disease) population, this thesis identifies 31 distinct single nucleotide polymorphisms within the hIP receptor, 17 non-synonymous (change in coding amino acid) and 14 synonymous (no change in amino acid). An R212C receptor variant was observed in 2.8% of diseased individuals. Furthermore, similar to the R212H polymorph, functional defects in agonist-induced receptor activation, binding affinity, and expression were observed in platelet-characterized receptors, derived from patient whole-blood samples. (7) From the same screening analysis, this thesis also examines the functional effects of several other naturally-occurring arginine-to-cysteine receptor variants, including R215C and R279C, and their effects on agonist-induced receptor activation, binding affinity, and expression. Again, receptors were characterized using patient whole-blood samples. |
