Characterization of the molecular motions of constitutively active G protein-coupled receptors for parathyroid hormone
Introduction
Parathyroid hormone (PTH) plays a pivotal role in the regulation of extracellular calcium homeostasis [1], [2]. PTH, along with vitamin D, is responsible for maintenance of serum calcium levels required for cellular function, adjusting for the variations from bone remodeling, renal function, and dietary intake of calcium. These physiological actions are mediated through the activation of the PTH receptor, PTH1, a member of the G protein-coupled receptor (GPCR) super family of heptahelical, transmembrane signaling proteins [3], [4].
In 1996, Jüppner and co-workers [5], [6] reported two single point mutations, H223R and T410P, in PTH1 which led to constitutively active receptors, ligand independent activity enhanced compared to basal levels. These mutations are related to Jansen's Metaphyseal Chondrodysplasia, a rare disease associated with hypercalcemia [5], [6], [7]. More recently, a third single point mutation, I458R, was observed in a patient with Jansen's disease [7], [8]. It has been shown that in mice the presence of the constitutively active receptor can overcome the effects of absence of PTH [9]. In addition to their medicinal relevance and usefulness as biochemical tools, the constitutively active receptors provide unique information into the molecular mechanism of receptor activation.
Here, we characterize the consequences of the three naturally occurring single point mutations (H223R, T410P, I458R) in the human PTH1 receptor using extensive molecular dynamics (MD) simulations. The simulations were carried out using a novel membrane mimetic which allows for extensive calculations while maintaining the overall, biphasic character of the membrane environment [10], [11]. From analysis of the simulations following established procedures [12], we correlate the single point mutations to short and long range effects on the receptor structure. Despite being dispersed through out the receptor in transmembrane helices (TM) 2, 6, and 7, (see Fig. 1), all of these substitutions lead to similar changes in the third cytoplasmic loop (IC3), centered on K405. Coupling these results with the high-resolution structure of IC3 of PTH1 [13], [14], provides insight into the molecular mechanism of ligand-independent receptor activation.
Section snippets
Model building
Modeling of the transmembrane (TM) domain of the human PTH receptor (accession number: Q03431) was carried out with the WHATIF program [15] as described previously [11], [16]. The method utilizes rhodopsin and bacteriorhodopsin [17], [18] as templates for the topological orientation and arrangement of the seven membrane-spanning helices. The receptor model was completed by addition of loops, generated using a metric matrix based DG program employing distances between adjacent TM helices as
Results
For a number of GPCRs, the extracellular N-terminus of the receptor is not required for the constitutive activity [27], [28], [29]. Our analysis of the native and mutant receptors of PTH1 is therefore focused on the relative motions of the TM helices and characterizing the effects these motions have on the intracellular domains. Utilizing procedures outlined by Weinstein and co-workers [12], the collective motions of the TM helices in the wild type and mutant receptors are compared and the
Discussion
From the first report of a constitutively active GPCR, it was recognized as a unique vehicle to probe the mechanism of receptor activation [31], [32], [33]. Many of the sites originally found to produce constitutive activity are located in the cytoplasmic portion of the receptor, especially prevalent in the third cytoplasmic loop [31], [32], [34], [35], [36], [37]. Not surprisingly IC3 has been shown to be strongly associated with coupling to the G protein [37], [38], [39], [40], [41], [42].
Acknowledgements
This work was supported, in part, by grant R29-GM54082 from the National Institutes of Health (D.F.M.) and grant 98/02903 from the Deutscher Akademischer Austauschdienst (C.R.). The authors wish to thank Dr Maria Pellegrini (Brown University) for advice and assistance.
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