Mapping the Bimolecular Interface of the Parathyroid Hormone (PTH)−PTH1 Receptor Complex:  Spatial Proximity between Lys<sup>27</sup>(of the Hormone Principal Binding Domain) and Leu<sup>261</sup>(of the First Extracellular Loop) of the Human PTH1 Receptor

Greenberg, Zvi; Bisello, Alessandro; Mierke, Dale F.; A, Rosenblatt; Chorev, Michael

doi:10.1021/bi000195n

Cited by 75 publications

(109 citation statements)

References 62 publications

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“…Purified PTH1R Have Functional N-terminal and Juxtamembrane Binding Domains-Binding of PTH and PTHrP to the PTH1R is consistent with a "two-site" model in which the C-terminal portion of the ligands interacts with the N-terminal ectodomain of the receptor, and N-terminal portion of the ligands binds to juxtamembrane domains (13,16,17,(37)(38)(39)(40)89). We therefore sought to establish the integrity of these apparently distinct binding domains by assessing competition between 125 I-PTH-(1-34) and four PTH or PTHrP fragments, PTH-(1-34), PTHrP-(5-36), Aib-PTH-(1-21), and Aib-PTH- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), whose binding properties with wild type hPTH1R has been characterized previously (89) on transfected intact COS-7 cells.…”

Section: Resultssupporting

confidence: 65%

“…Understanding hormone-PTH1R and PTH1R-G protein interactions has relied largely on determination of functional consequences resulting from mutations in either the hormone or the receptor (13,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), analysis of receptor fragments after cross-linking to radioiodinated, p-benzoyl-L-phenylalaninemodified ligands (13,(37)(38)(39)(40), crystallographic resolution of PTH (41), and NMR of PTH (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), PTHrP (59 -68), and short segments of the PTH1R (see Refs. 69 and 70 and for review see Refs.…”

Section: The Parathyroid Hormone (Pth)mentioning

confidence: 99%

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Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Shimada

Chen

Cvrk

et al. 2002

Journal of Biological Chemistry

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The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 g of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr ]GTP␥S incorporation into G␣ s in a time-and dose-dependent manner, when recombinant hPTH1R, G␣ s -, and ␤␥-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.

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Section: Resultssupporting

confidence: 65%

Section: The Parathyroid Hormone (Pth)mentioning

confidence: 99%

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Shimada

Chen

Cvrk

et al. 2002

Journal of Biological Chemistry

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“…The Ser1 residue used in the simulations adopts a position equidistant between the extracellular portion of TM5 and TM6, consistent with the general mode of GPCR activation by ligand. (4)(5)(6)8,9,12). By combining data collected from extensive photoaffinity cross-linking studies with molecular modeling, a model of the PTH-PTHR1 bimolecular complex has been generated that identifies regions of contact at the interface between the hormone and the receptor.…”

Section: Molecular Modeling Of the Site Of Interaction Of Position 1 mentioning

confidence: 99%

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

et al. 2008

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Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine-(Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH-PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH-PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH-PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide-receptor interfaces and should yield insights about yet unknown sites of ligand-receptor interactions, allowing for generation of more refined models.Class II G protein-coupled receptors (GPCRs) 1 interact with physiologically important peptides. There is intense study of how these peptides associate with their cognate receptors since elucidation of these interactions should provide important insights for the rational design of ligands with enhanced pharmacological properties for use in treating an array of diseases (1,2). Over the past decade, photoaffinity cross-linking has been employed to study the interaction of a number of peptide-GPCR interactions (3). This technique involves systematically probing the receptor for regions of interaction using a peptide that incorporates a photoreactive moiety that irreversibly cross-links to the receptor. We and others have used this approach extensively to study the interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1) (4-12). This hormone-receptor system plays an integral role in calcium metabolism and bone biology. † This work was supported in part by Grants DK47490 (to M.R.) and GM54082 (to D.F.M.) from the National Institutes of Health.* Address correspondence to this author. Phone: (617) Our research is focused on studying the bimolecular interface of the PTH-PTHR1 complex in order to gain insights that will aid the design of ligands of PTHR1 for the treatment of osteoporosis and o...

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“…It is also similar to covalent labeling of the distal amino-terminal tail of the PTH 1 receptor by PTH/PTHrP probes, incorporating a photolabile residue in positions 23 (47) and 28 (46) (Table I). The identification of the amino-terminal domain of the calcitonin receptor as the labeling domain for both Bpa 16 and Bpa 26 probes is distinct from photoaffinity labeling of the first extracellular loop domain of the PTH 1 receptor by a position 27 probe (51) and from that of the top of the sixth transmembrane domains of the PTH 1 receptor by PTH/PTHrP probes incorporating a photolabile residue at their amino termini (52,53) (Table I).…”

Section: Identification Of Domains Of Labeling By Peptidementioning

confidence: 99%

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

Dong¹,

Pinon²,

Cox

et al. 2004

Journal of Biological Chemistry

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The calcitonin receptor is a member of the class B family of G protein-coupled receptors, closely related to secretin and parathyroid hormone receptors. Although mechanisms of ligand binding have been directly explored for those receptors, current knowledge of the molecular basis of calcitonin binding to its receptor is based only on receptor mutagenesis. In this work we have utilized the more direct approach of photoaffinity labeling to explore spatial approximations between distinct residues within calcitonin and its receptor. For this we have developed two human calcitonin analogues incorporating a photolabile p-benzoyl-L-phenylalanine residue in the mid-region and carboxyl-terminal half of the peptide in positions 16 and 26, respectively. Both probes specifically bound to the human calcitonin receptor with high affinity and were potent stimulants of cAMP accumulation in calcitonin receptor-bearing human embryonic kidney 293 cells. They covalently labeled the calcitonin receptor in a saturable and specific manner. Further purification, deglycosylation, specific chemical and enzymatic cleavage, and sequencing of labeled wild type and mutant calcitonin receptors identified the sites of labeling for the position 16 and 26 probes as receptor residues Phe 137 and Thr 30 , respectively. Both were within the extracellular amino terminus of the calcitonin receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor. These data are consistent with affinity labeling of other members of the class B G protein-coupled receptors using analogous probes and may suggest a common ligand binding mechanism for this family.Calcitonin, a hypocalcemic peptide hormone, is secreted from the thyroid gland in response to elevations in serum calcium levels. Its hypocalcemic effect is mediated by inhibition of bone resorption by osteoclasts and enhancement of renal calcium excretion. These actions are important for its widespread clinical use for treatment of bone disorders, including Paget's disease, osteoporosis, and hypercalcemia of malignancy (1, 2).The calcitonin peptide consists of 32 amino acids, with a disulfide bond between residues 1 and 7 which is conserved among all species and that is believed to be critical for its agonist activity. The amino acid sequence and biological potency of calcitonin vary considerably from species to species, but the integrity of the disulfide bond and the carboxyl-terminal proline-amide are necessary for full biological activity (1-3). The disulfide bond can be replaced by other covalent bonds that lead to improved biological stability while retaining full potency (1). The sequence within the amino-terminal loop region is highly conserved in a variety of species but demonstrates divergence in the rest of the sequence (4). Like secretin and peptides for other members of class B G protein-coupled receptor family, the amino-terminal region of calcitonin contains key determinants for receptor agonist selectivity, whereas the...

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Mapping the Bimolecular Interface of the Parathyroid Hormone (PTH)−PTH1 Receptor Complex: Spatial Proximity between Lys²⁷(of the Hormone Principal Binding Domain) and Leu²⁶¹(of the First Extracellular Loop) of the Human PTH1 Receptor

Cited by 75 publications

References 62 publications

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

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Mapping the Bimolecular Interface of the Parathyroid Hormone (PTH)−PTH1 Receptor Complex: Spatial Proximity between Lys27(of the Hormone Principal Binding Domain) and Leu261(of the First Extracellular Loop) of the Human PTH1 Receptor

Cited by 75 publications

References 62 publications

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

Mapping Peptide Hormone−Receptor Interactions Using a Disulfide-Trapping Approach

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

Contact Info

Product

Resources

About

Mapping the Bimolecular Interface of the Parathyroid Hormone (PTH)−PTH1 Receptor Complex: Spatial Proximity between Lys²⁷(of the Hormone Principal Binding Domain) and Leu²⁶¹(of the First Extracellular Loop) of the Human PTH1 Receptor