Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor

For many G protein-coupled receptors, agonist-induced activation is followed by desensitization, internalization, and resensitization. In most cases, these processes are dependent upon interaction of agonistoccupied receptor with cytoplasmic ␤-arrestins. The ligand-induced intramolecular rearrangements of the receptor responsible for the desensitized versus active conformational states, which dictate both the pharmacological properties of ligands and the biological activity of G protein-coupled receptors, have not been fully elucidated. Here, we identify specific interactions between parathyroid hormone (PTH)-related protein and the human PTH type 1 receptor (PTH1Rc) and the related receptor conformational changes that lead to ␤-arrestin-2-mediated desensitization. PTH-related protein analogs modified at position 1 induced selective stabilization of the active G protein-coupled state of the receptor, resulting in lack of ␤-arrestin-2 recruitment to the cell membrane, sustained cAMP signaling, and absence of ligand-receptor complex internalization. Mechanistically, the ligands modified at position 1, interacting with the extracellular end of helix VI of PTH1Rc, produced a translocation of transmembrane helices V and VI that differed from that induced by the cognate agonist, resulting in significantly different conformations of the third intracellular loop. These results show how specific interactions between PTH1Rc and its ligands may stabilize distinct conformational states, representing either the active G protein-coupled or a desensitized ␤-arrestin-coupled receptor state. In addition, they establish that sustained biological activity of PTH1Rc may be induced by appropriately designed agonist ligands. G protein-coupled receptors (GPCRs)1 represent a major class of membrane-bound proteins that mediate a wide variety of biological functions, including sensitivity to light and odorants, endocrine and cardiovascular control, and neurotransmission. The ligand-induced intracellular signaling of GPCRs is tightly regulated by several mechanisms. For numerous GPCRs, desensitization involves translocation of arrestins from the cytosol to the cell membrane, their direct interaction with agonist-activated GPCRs, and consequent inhibition of G protein coupling (1, 2). Additionally, arrestin-mediated internalization of agonist-receptor complexes through clathrincoated vesicles and subsequent receptor recycling to the cell membrane are responsible for the recovery of cellular responsiveness to agonists (resensitization). Receptor desensitization and resensitization accomplish the fundamental physiological role of modulating the cellular responses to both acute and chronic stimulation. An important implication is that not only signal transduction per se, but also the mechanisms regulating signal transduction have a profound influence in determining the pathophysiological processes mediated by GPCRs. Many important questions remain regarding the relationship between receptor occupancy, signaling, and desensitization. In partic...

Section: Characterization Of Position 1-modified Pthrp Analogs-supporting

confidence: 84%

Selective Ligand-induced Stabilization of Active and Desensitized Parathyroid Hormone Type 1 Receptor Conformations

Bisello¹,

Chorev²,

Rosenblatt³

et al. 2002

“…Consistent with these mutational data, Bpa introduced at position 1 of PTH- or position 2 of either PTH- 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)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(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 (12).…”

supporting

confidence: 65%

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

Self Cite

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 ...

“…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%

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.