Versatility and Differential Roles of Cysteine Residues in Human Prostacyclin Receptor Structure and Function

Jeremiah Stitham,John Hwa,Eric Arehart,Karen Douville,Scott R. Gleim

doi:10.1074/jbc.m604042200

Abstract

Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).

Highlights

Cysteine chemistry is both fascinating and intriguing
Related to the severe binding defects, potency (EC50) values could not be determined due to negligible efficacy (Table 1). These results demonstrate the severity of functional perturbation with mutation of any one of the four extracellular cysteine residues within the human prostacyclin receptor (hIP) and lend preliminary support to the notion that 1) the highly conserved Cys-92 and Cys-170 may form a disulfide bond (Fig. 1B), and 2) an alternative interaction may exist between the remaining extracellular cysteines Cys-5 and Cys165, which appears to be important for hIP receptor binding and activation
Prostacyclin (PGI2) analogs are widely used for the treatment of pulmonary hypertension (30 –33)

Summary

Introduction

Cysteine chemistry is both fascinating and intriguing. The sulfhydryl or thiol (SH) reactive groups of this amino acid are very susceptible to oxidation and can readily form stable dimers (i.e. disulfide S-S bridges), which play important roles in the organization and maintenance of protein tertiary structure. We report that in addition to the highly conserved disulfide interaction between Cys-92 (top of transmembrane (TM) III) and Cys-170 (exoloop 2), an additional, putative non-conserved disulfide bridge may exist between Cys-5 (N terminus) and Cys-165 (exoloop 2), within the extracellular domain of the hIP, as well as other prostanoid receptors (hDP and hEP2). In the transmembrane domain Cys-118 (TMIII), Cys-202 (TMV), and Cys-251 (TMVI) were found to be necessary for preserving normal receptor binding affinity, activation capacity, and/or cell-surface expression through probable hydrogen bonding networks Such observations provide further insights into the molecular functioning of the hIP receptor and contribute to the mechanistic understanding of how the hIP protein is stabilized during the continuum of conformational changes that occur upon agonist-induced activation

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Conclusion