Research Areas

Prostacyclin Receptor

Prostacyclin Receptor Project:

"Why are prostacyclin and the prostacyclin receptor so important?"

1. Prostacyclin (PGI2) is an endogenous factor that exerts cardioprotective effects, such as inhibition of platelet aggregation, and is crucial for maintaining vascular homeostasis.

2. Studies using prostacyclin receptor knock-out (IP-/-) mice have demonstrated animals with enhanced thrombotic response to vascular injury, increased incidence of restenosis, and propensity towards atherogenesis.

3. The world-wide withdrawal of the selective COX-2 inhibitor, Vioxx(R) (rofecoxib), has been linked to a drug-induced prostacyclin (PGI2) deficiency that increases the risk of hazardous cardiovascular events in human subjects.

Structure-Function Studies:

Despite its importance, very little is known about how the hIP receptor functions at the molecular level, interacting with ligand and propagating signal through its transmembrane (TM) domain to activate intracellular signaling molecules. Our lab uses site-directed (PCR) mutagenesis, computer-assisted homology modeling, and functional analyses to gain critical insights into the molecular mechanisms of both agonist binding and receptor activation. We have generated a 3-D homology model of the hIP transmembrane domain (based upon the 2.8A crystal structure of rhodopsin).

Pharmacogenetics (POP Trial) Studies:

Clinical trials have shown that selective suppression of COX-2-derived prostacyclin (PGI2) increases the risk of adverse cardiovascular events (e.g., myocardial infarction and thrombotic stroke), particularly in patients predisposed to cardiovascular disease. Such effects, based upon deficiencies in COX-2-mediated ligand production, prompted us to look further down the biosynthetic-signaling pathway --- at the level of the receptor --- with the notion that a functionally-defective hIP receptor (decreased activity due to a polymorphism within the PTGIR gene) might have a similar effect as decreased amounts of ligand (as seen with selective COX-2 inhibition), resulting in increased cardiovascular events. Our group has undertaken a large-scale genetic screening study, Pharmacogenetics of Prostacyclin (POP) Trial, searching for single-nucleotide polymorphisms (SNPs) and mutations within the hIP receptor gene that could result in a defective protein.

Biosynthetic and Signaling Pathways Studies:

Endothelial cells are considered to be the main source of prostacyclin (PGI2) within the vascular system, while platelets and vascular smooth muscle cells (VSMCs) serve as the predominate sites for prostacyclin receptor (IP) expression. In collaboration with our colleagues in the laboratory of Dr. Kathleen Martin, Department of Vascular Surgery, we are actively investigating both the biosynthetic and signaling pathways in human VSMCs (hVSMCs), in order to discern the role of endogenous prostacyclin in theses cells. Studies using primary vascular smooth muscle cells from patient donors have shown that prostacyclin (PGI2) may act, in both a paracrine and autocrine fashion, to modulate hVSMC activity and may play a significant role in the prevention of both atherosclerosis (cardioprotective) and carcinogenesis.

Structure-Function

Transmembrane Prolines (PubMed)
Binding Pocket (PubMed)
Activation Clusters (PubMed)

Pharmacogenetics

V25M & R212H Polymorphisms (PubMed)

Rhodopsin

Rhodopsin, Zinc & Retinitis Pigmentosa studies

The laboratory also studies structure and function relationships in another G-protein coupled receptor, rhodopsin. Missense mutations or short in-frame deletions in the gene encoding the dim light photoreceptor rhodopsin can lead to Retinitis Pigmentosa (RP), a debilitating inherited disease characterized by retinal degeneration and progressive blindness. Using protein chemistry and biophysical techniques, Dr. Hwa's laboratory is exploring the structural perturbations that give rise to the RP rhodopsin misfolding. These studies have revealed common structural defects amongst many of the misfolded RP proteins including disrupted interhelical interactions, an inability to bind 11-cis retinal (vitamin A analogue), and the formation of an abnormal disulfide bond. Furthermore, there are intriguing interactions with trace metals. Transmembrane zinc binding promotes rhodopsin stability. In contrast, extracellular zinc binding leads to rhodopsin destabilization. Such interactions may play critical roles in the pathogenesis of retinal degeneration in RP. The laboratory is currently exploring such defects as potential targets for therapy

Structure-Function

Rhodopsin and Retinitis Pigmentosa (PubMed)

Zinc Coordination & RP

Rhodopsin Zinc Binding & RP (PubMed)
RP L125R and A164V (PubMed)