Bioactivities and Secondary Structures of Constrained Analogues of Human Parathyroid Hormone:  Cyclic Lactams of the Receptor Binding Region

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/ PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [ -172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex. PTH and PTHrP1 mediate many of their biological effects through the same receptor (1, 2). Peptides containing the first 34 amino acids of PTH and PTHrP are capable of fully activating the PTH/PTHrP receptor (P1R) (1, 2). Studies with PTH and PTHrP ligand analogs and receptor chimeras have suggested that the ligands have two distinct functional domains: the amino-terminal residues, which are important for receptor activation; and the carboxyl-terminal residues, which are important for high affinity binding (3). These data furthermore indicate that the ligand's amino-terminal portion interacts with the extracellular loops and the membrane-spanning helices of the receptor, and the ligand's carboxyl-terminal portion interacts with the receptor's amino-terminal extracellular domain (3). A similar pattern of ligand-receptor interaction has been suggested for other members of this class II family of peptide hormone G-protein-coupled receptors, including the secretin receptor (4) and the PTH-2 receptor (5-7).Some specific sites of interaction between the amino-terminal portions of PTH-(1-34)/PTHrP-(1-36) and the P1R have been identified by site-directed mutagenesis and photoaffinity cross-linking studies. For example, mutational analyses have shown that residues in extracellular loop 3 and the adjacent sixth membrane-spanning helix (TM6) are critical for mediating ligand-induced receptor activation (2,8). Consistent with these mutational data, Bpa introduced at position 1 of PTH-(1-34) or position 2 of either PTH-(1-34) or PTHrP-(1-36) was found to cross-link to methionine 425, at the extracellular end of TM6 in the P1R (9, 10). Interactions between the aminoterminal portion of the ligand and the transmembrane domains/extracellular loops of the receptor are also suggested by a study showing that PTH-(1-14) can stimulate cAMP accumulation in a mutant P1R missing most of the amino-terminal extracellular domain as efficiently as it does in the wild-type P1R (11). Furthermore, if a PTH fragment comprising the first 9 amino acids is covalently attached to the amino-terminal end of such a truncated receptor, the resulting ligand-receptor chimera displays constitutive activity, indicative of an intramolecular stimulation of the receptor's activation domain by the tethered ligand fragment ...

Section: Analogsmentioning

confidence: 90%

Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1–36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking

Gensure¹,

Gardella²,

Jüppner³

2001

“…Peptides were synthesized using an Fmoc protocol as described previously (32). Fmoc-␣-methyl-Leu and Fmoc-␣-methyl-Asp were purchased from Chem-Impex and Fmoc-␣-methyl-Val from Novabiochem (La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone

Barbier

Gardella

Dean

et al. 2005

Self Cite

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic ␣-helix of PTH (1-31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal ␣-helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an ␣-helix. Binding and adenylyl cyclase-stimulating data showed that outside of the ␣-helix, methylation of residues Asp 30 and Val 31 had little effect on structure or activities. Within the ␣-helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val 21 , Leu 24 , Arg 25 , and Leu 28 there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser 17 , Trp 23 , and Lys 26 as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.

“…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%

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

Shimada

Chen

Cvrk

et al. 2002

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.