Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor (2001)

Ligand binding to the PTH1 receptor is described by a “two-site” model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3–34) and PTH(1–14): PTH(1–14), which binds predominantly to the J domain, only partially inhibited binding of125I-PTH(3–34); and PTH(3–34), shown to bind predominantly to the N domain, only partially inhibited PTH(1–14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of125I-PTH(3–34) with 30 μm guanosine 5′-3-O-(thio)triphosphate (GTPγS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1–36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1–34) affinity for RG; residue 5 of MAP-PTHrP(1–36) was a determinant of R/RG binding selectivity, and PTH(1–14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3–34) binding was GTPγS-insensitive. Finally, several observations suggest the receptor conformation is more “closed” at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1–14) and PTH(3–34). At RG PTH(1–14) better occluded binding of 125I-PTH(3–34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states. Ligand binding to the PTH1 receptor is described by a “two-site” model, in which the C-terminal portion of the ligand interacts with the N-terminal domain of the receptor (N interaction), and the N-terminal region of the ligand binds the juxtamembrane domain of the receptor (J interaction). Previous studies have not considered the dynamic nature of receptor conformation in ligand binding and receptor activation. In this study the ligand binding mechanism was compared for the G-protein-coupled (RG) and uncoupled (R) PTH1 receptor conformations. The two-site model was confirmed by demonstration of spatially distinct binding sites for PTH(3–34) and PTH(1–14): PTH(1–14), which binds predominantly to the J domain, only partially inhibited binding of125I-PTH(3–34); and PTH(3–34), shown to bind predominantly to the N domain, only partially inhibited PTH(1–14)-stimulated cAMP accumulation. To assess the effect of R-G coupling, ligand binding to R was measured by displacement of125I-PTH(3–34) with 30 μm guanosine 5′-3-O-(thio)triphosphate (GTPγS) present, and binding to RG was measured by displacement of 125I-[MAP]PTHrP(1–36) (where MAP is model amphipathic peptide), a new radioligand that binds selectively to RG. Agonists bound with higher affinity to RG than R, whereas antagonists bound similarly to these states. The J interaction was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1–34) affinity for RG; residue 5 of MAP-PTHrP(1–36) was a determinant of R/RG binding selectivity, and PTH(1–14) bound selectively to RG. The N interaction was insensitive to R-G coupling; PTH(3–34) binding was GTPγS-insensitive. Finally, several observations suggest the receptor conformation is more “closed” at RG than R. At the R state, an open conformation is suggested by the simultaneous binding of PTH(1–14) and PTH(3–34). At RG PTH(1–14) better occluded binding of 125I-PTH(3–34) and agonist ligands bound pseudo-irreversibly, suggesting a more closed conformation of this receptor state. The results extend the two-site model to take into account R and RG conformations and suggest a model for differences of receptor conformation between these states. parathyroid hormone PTH-related protein bovine human rat model amphipathic peptide tuberoinfundibular peptide (7–39) G-protein G-protein-coupled receptor G-protein-uncoupled receptor state G-protein-coupled receptor state guanosine 5′-2-O-(thio)diphosphate guanosine 5′-3-O-(thio)triphosphate The parathyroid hormone 1 (PTH1)1 receptor is a cell-surface signal transducer for PTH and PTH-related protein (PTHrP). PTH plays a central role in calcium homeostasis; the hormone acts on target cells in bone (osteoblasts) and kidney (renal tubule cells) to increase blood calcium levels (1Potts Jr., J.T. Bringhurst F.R. Gardella T. Nussbaum S. Segre G. Kronenberg H. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1995: 920-966Google Scholar). PTHrP is an autocrine factor, believed to be involved in the maintenance of numerous tissues, and an important developmental regulator, controlling breast, pancreas, skin, and bone development (2Martin T.J. Moseley J.M. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1995: 967-977Google Scholar, 3Wysolmerski J.J. Stewart A.F. Annu. Rev. Physiol. 1998; 60: 431-460Crossref Scholar). PTH and PTHrP involved in the and of acts a bone for the of T.J. Scholar). PTHrP is by to of the PTH1 receptor W. T.J. 1998; Scholar). The by PTH and PTHrP the PTH1 receptor of of and of and H. T. Jr., J.T. Kronenberg Segre Scholar, Scholar, F.R. H. Jr., J.T. Kronenberg Segre Endocrinology. Scholar, Endocrinology. Scholar, Endocrinology. to important and the of PTH1 receptor have H. Gardella T.J. Physiol. Scholar). The receptor to the of G-protein-coupled which to peptide ligands of and The receptor be into the N-terminal domain (N to of the binding for interaction Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. and the juxtamembrane region of the receptor (J is a determinant of receptor and Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. Scholar, T.J. H. Wilson Segre Bringhurst F.R. Jr., J.T. Nussbaum Kronenberg Endocrinology. Scholar, T. Scholar). the ligand be into binding the portion is a determinant of receptor binding affinity Gardella T.J. Jr., J.T. Kronenberg H. Scholar, J.J. Endocrinology. Scholar, Segre Nussbaum Jr., J.T. Endocrinology. and the portion is a determinant of receptor for of cAMP Gardella T.J. Jr., J.T. Kronenberg H. Scholar, T.J. Jr., J.T. Kronenberg Nussbaum Scholar, Gardella T.J. Scholar, Jr., J.T. Scholar, Jr., J.T. Gardella T.J. Scholar). and affinity binding of PTH and PTHrP an N-terminal of residues Jr., J.T. R. S. observations suggested a “two-site” of interaction in which the C-terminal portion of the ligand interacts with the N domain of the receptor (N interaction), and the N-terminal ligand region binds to the J domain of the receptor (J H. Gardella T.J. Physiol. Scholar, Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. Scholar, Gardella T.J. Scholar). model for S. R. Scholar, Scholar, Scholar, Scholar). the PTH1 this model is by a of receptor and which have suggested of between of the ligand and the receptor T.J. H. Wilson Segre Bringhurst F.R. Jr., J.T. Nussbaum Kronenberg Endocrinology. Scholar, T. Scholar, 1998; Scholar, 1998; T.J. Scholar, Scholar, Gardella T.J. H. 1998; Scholar). observations have with ligand S. Scholar, 1998; and of the receptor to of of interaction 1998; Scholar, S. Scholar, Scholar, interaction for the PTH1 receptor have not into account the dynamic nature of receptor is central to the of a to the signal of ligand binding the to the signal of G-protein 1998; Scholar, S. T. Scholar). a the effect of this receptor is for ligand binding and signal 1998; Scholar, S. T. Scholar). have for of the the to the development of the model and 1998; Scholar, S. T. Scholar, J.M. Scholar). the of G-protein and agonist on binding to the receptor a is the of ligand is the two-site binding mechanism is by the in the receptor that R-G In this study the effect of R-G interaction on the two-site binding mechanism for the PTH1 studies and ligands have a two-site of ligand by the PTH1 The C-terminal portion of the of PTH PTHrP interacts with the N-terminal receptor region (N interaction), and the N-terminal portion of the ligand binds to the juxtamembrane domain of the receptor (J H. Gardella T.J. Physiol. Scholar, Gardella T.J. Jr., J.T. Kronenberg H. Scholar). In this study the effect of receptor interaction on the mechanism of ligand binding to the PTH1 The of this study interaction for the receptor is described by the two-site for this binding was by the of between the binding of PTH(1–14) and PTH(3–34) to the receptor with the of simultaneous receptor binding of these ligands bind with higher affinity to the RG state than to the uncoupled whereas ligands bind with affinity to these states. The J interaction is by R-G coupling, whereas the N interaction is not by R-G more closed receptor conformation is suggested for the PTH1 receptor to in the model in for of ligand binding to the PTH1 receptor by the C-terminal portion of the ligand interacts with the N domain of the the N-terminal portion of the ligand binds to the J domain of the receptor R-G interaction the affinity of the J by a of the receptor interaction of the ligand with the J domain the affinity of receptor for G-protein activation. of G-protein to the of the receptor and for of between the binding of PTH(1–14) and PTH(3–34) the two-site model for the PTH1 The of simultaneous binding of PTH(1–14) and PTH(3–34) is with and of sites that with the and of the Gardella T.J. Jr., J.T. Kronenberg H. Scholar). The into the mechanism of PTH(3–34) PTH(1–14), to of PTH(3–34) with the juxtamembrane domain Jr., J.T. Gardella T.J. effect on the receptor binding affinity of 125I-PTH(3–34) increase of at the G-protein-uncoupled that PTH(3–34) not with the juxtamembrane domain, that the binding for PTH(3–34) is by the N effect of on ligand binding to the receptor was by ligand affinity for the uncoupled receptor with the affinity for the RG The was measured by displacement of 125I-PTH(3–34) binding in the of 30 μm the a new radioligand that selectively the RG an of the bone T. Scholar). The R/RG of this radioligand was by the of binding by which the RG Annu. Rev. and by the increase of binding by of the PTH1 receptor with a that the RG state agonist ligands bound with higher affinity to the RG than to the uncoupled whereas ligands bound with affinity to these the of studies of the PTH1 receptor G. Endocrinology. and in with a of studies of S. T. Scholar). that G-protein by the RG state. of ligand binding to R and RG for a of to of In this study of the of ligand affinity for R and RG and the of 125I-[MAP]PTHrP(1–36) binding with affinity by the ligand The binding in this study be for by the of the model, was between these J.M. Scholar). of the receptor the and of the receptor in the model S. T. of the G-protein in with the to the the role of the G-protein in agonist binding to the PTH1 receptor to be the not agonist binding is required with the for the of the affinity agonist binding state Scholar, H. the of agonist ligands was increased by R-G the to which the the two-site binding mechanism N were by R-G of the N-terminal region of the ligand the binding of the N-terminal residues PTH(1–34) in the of of of with the to a role for residue 5 in the of binding suggest that the affinity of ligand interaction with the J domain is increased by RG a by the of binding of PTH(1–14) to the RG state. In ligand binding to the N domain is not to RG coupling; PTH(3–34) bound with affinity to the R and RG of the R-G interaction ligand interaction with the J domain and not ligand binding to the N domain increase of ligand affinity for RG compared with R receptor conformations at the G-protein-coupled and receptor states. At the uncoupled the binding suggest than the conformation is open to the simultaneous binding of PTH(1–14) and PTH(3–34). the RG state, observations with the of a more closed receptor binding is suggesting that the ligand is the G. Scholar). PTH(1–14) a of the binding affinity of PTH(3–34) at the RG state, suggesting that simultaneous binding of ligand better binding of the PTH(3–34) of PTH(1–14)-stimulated cAMP accumulation. a of the PTH1 receptor to an conformation that to PTH(3–34) and PTH(1–14) bound to the The open and closed in this of the open and closed an is the of the The on of receptor conformation binding more this study on the conformation of the the conformation of the ligand bound to the receptor is the a of PTH(1–34) an conformation S. Scholar). studies of PTH and PTHrP with an in the and C-terminal by a region of 1998; Scholar, S. S. Scholar). bound to the receptor studies suggest an ligand conformation S. whereas studies suggest between the and C-terminal ligand T.J. Wilson Nussbaum Jr., J.T. Kronenberg Scholar). In this a of the binding of PTH(3–34) and is to with the conformation binds with affinity to the PTH1 receptor than PTH(3–34). the effect of PTH(1–14) binding on the affinity of PTH(3–34) that of the binding of PTH(3–34) is by receptor the portion of the a the of affinity of is to by the of a interaction of this region of the ligand with the juxtamembrane region of the The effect be for by the of the which in the of affinity of the ligand region with the for the have the two-site model to take into account conformations of the PTH1 the R and RG states. ligand interaction with the J domain of the PTH1 receptor the RG state the R state suggesting that the J interaction interaction and Ligand binding to the N domain is insensitive to R-G Finally, the increase of agonist affinity for the RG state a of the receptor the of the binding mechanism for these be to an of the signal mechanism of of this receptor The parathyroid hormone 1 (PTH1)1 receptor is a cell-surface signal transducer for PTH and PTH-related protein (PTHrP). PTH plays a central role in calcium homeostasis; the hormone acts on target cells in bone (osteoblasts) and kidney (renal tubule cells) to increase blood calcium levels (1Potts Jr., J.T. Bringhurst F.R. Gardella T. Nussbaum S. Segre G. Kronenberg H. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1995: 920-966Google Scholar). PTHrP is an autocrine factor, believed to be involved in the maintenance of numerous tissues, and an important developmental regulator, controlling breast, pancreas, skin, and bone development (2Martin T.J. Moseley J.M. Williams R.H. Wilson J.D. Foster D.W. Williams' Textbook of Endocrinology. W. B. Saunders Co., Philadelphia1995: 967-977Google Scholar, 3Wysolmerski J.J. Stewart A.F. Annu. Rev. Physiol. 1998; 60: 431-460Crossref Scholar). PTH and PTHrP involved in the and of acts a bone for the of T.J. Scholar). PTHrP is by to of the PTH1 receptor W. T.J. 1998; Scholar). The by PTH and PTHrP the PTH1 receptor of of and of and H. T. Jr., J.T. Kronenberg Segre Scholar, Scholar, F.R. H. Jr., J.T. Kronenberg Segre Endocrinology. Scholar, Endocrinology. Scholar, Endocrinology. Scholar). to important and the of PTH1 receptor have H. Gardella T.J. Physiol. Scholar). The receptor to the of G-protein-coupled which to peptide ligands of and The receptor be into the N-terminal domain (N to of the binding for interaction Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. and the juxtamembrane region of the receptor (J is a determinant of receptor and Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. Scholar, T.J. H. Wilson Segre Bringhurst F.R. Jr., J.T. Nussbaum Kronenberg Endocrinology. Scholar, T. Scholar). the ligand be into binding the portion is a determinant of receptor binding affinity Gardella T.J. Jr., J.T. Kronenberg H. Scholar, J.J. Endocrinology. Scholar, Segre Nussbaum Jr., J.T. Endocrinology. and the portion is a determinant of receptor for of cAMP Gardella T.J. Jr., J.T. Kronenberg H. Scholar, T.J. Jr., J.T. Kronenberg Nussbaum Scholar, Gardella T.J. Scholar, Jr., J.T. Scholar, Jr., J.T. Gardella T.J. Scholar). and affinity binding of PTH and PTHrP an N-terminal of residues Jr., J.T. R. S. observations suggested a “two-site” of interaction in which the C-terminal portion of the ligand interacts with the N domain of the receptor (N interaction), and the N-terminal ligand region binds to the J domain of the receptor (J H. Gardella T.J. Physiol. Scholar, Gardella T.J. Jr., J.T. Kronenberg H. Scholar, H. Bringhurst F.R. Jr., J.T. Kronenberg Segre Gardella T.J. Endocrinology. Scholar, Gardella T.J. Scholar). model for S. R. Scholar, Scholar, Scholar, Scholar). the PTH1 this model is by a of receptor and which have suggested of between of the ligand and the receptor T.J. H. Wilson Segre Bringhurst F.R. Jr., J.T. Nussbaum Kronenberg Endocrinology. Scholar, T. Scholar, 1998; Scholar, 1998; T.J. Scholar, Scholar, Gardella T.J. H. 1998; Scholar). observations have with ligand S. Scholar, 1998; and of the receptor to of of interaction 1998; Scholar, S. Scholar, Scholar, Scholar). interaction for the PTH1 receptor have not into account the dynamic nature of receptor is central to the of a to the signal of ligand binding the to the signal of G-protein 1998; Scholar, S. T. Scholar). a the effect of this receptor is for ligand binding and signal 1998; Scholar, S. T. Scholar). have for of the the to the development of the model and 1998; Scholar, S. T. Scholar, J.M. Scholar). the of G-protein and agonist on binding to the receptor a is the of ligand is the two-site binding mechanism is by the in the receptor that R-G In this study the effect of R-G interaction on the two-site binding mechanism for the PTH1 studies and ligands have a two-site of ligand by the PTH1 The C-terminal portion of the of PTH PTHrP interacts with the N-terminal receptor region (N interaction), and the N-terminal portion of the ligand binds to the juxtamembrane domain of the receptor (J H. Gardella T.J. Physiol. Scholar, Gardella T.J. Jr., J.T. Kronenberg H. Scholar). In this study the effect of receptor interaction on the mechanism of ligand binding to the PTH1 The of this study interaction for the receptor is described by the two-site for this binding was by the of between the binding of PTH(1–14) and PTH(3–34) to the receptor with the of simultaneous receptor binding of these ligands bind with higher affinity to the RG state than to the uncoupled whereas ligands bind with affinity to these states. The J interaction is by R-G coupling, whereas the N interaction is not by R-G more closed receptor conformation is suggested for the PTH1 receptor to in the model in of between the binding of PTH(1–14) and PTH(3–34) the two-site model for the PTH1 The of simultaneous binding of PTH(1–14) and PTH(3–34) is with and of sites that with the and of the Gardella T.J. Jr., J.T. Kronenberg H. Scholar). The into the mechanism of PTH(3–34) PTH(1–14), to of PTH(3–34) with the juxtamembrane domain Jr., J.T. Gardella T.J. effect on the receptor binding affinity of 125I-PTH(3–34) increase of at the G-protein-uncoupled that PTH(3–34) not with the juxtamembrane domain, that the binding for PTH(3–34) is by the N effect of on ligand binding to the receptor was by ligand affinity for the uncoupled receptor with the affinity for the RG The was measured by displacement of 125I-PTH(3–34) binding in the of 30 μm the a new radioligand that selectively the RG an of the bone T. Scholar). The R/RG of this radioligand was by the of binding by which the RG Annu. Rev. and by the increase of binding by of the PTH1 receptor with a that the RG state agonist ligands bound with higher affinity to the RG than to the uncoupled whereas ligands bound with affinity to these the of studies of the PTH1 receptor G. Endocrinology. and in with a of studies of S. T. Scholar). that G-protein by the RG state. of ligand binding to R and RG for a of to of In this study of the of ligand affinity for R and RG and the of 125I-[MAP]PTHrP(1–36) binding with affinity by the ligand The binding in this study be for by the of the model, was between these J.M. Scholar). of the receptor the and of the receptor in the model S. T. of the G-protein in with the to the the role of the G-protein in agonist binding to the PTH1 receptor to be the not agonist binding is required with the for the of the affinity agonist binding state Scholar, H. the of agonist ligands was increased by R-G the to which the the two-site binding mechanism N were by R-G of the N-terminal region of the ligand the binding of the N-terminal residues PTH(1–34) in the of of of with the to a role for residue 5 in the of binding suggest that the affinity of ligand interaction with the J domain is increased by RG a by the of binding of PTH(1–14) to the RG state. In ligand binding to the N domain is not to RG coupling; PTH(3–34) bound with affinity to the R and RG of the R-G interaction ligand interaction with the J domain and not ligand binding to the N domain increase of ligand affinity for RG compared with R receptor conformations at the G-protein-coupled and receptor states. At the uncoupled the binding suggest than the conformation is open to the simultaneous binding of PTH(1–14) and PTH(3–34). the RG state, observations with the of a more closed receptor binding is suggesting that the ligand is the G. Scholar). PTH(1–14) a of the binding affinity of PTH(3–34) at the RG state, suggesting that simultaneous binding of ligand better binding of the PTH(3–34) of PTH(1–14)-stimulated cAMP accumulation. a of the PTH1 receptor to an conformation that to PTH(3–34) and PTH(1–14) bound to the The open and closed in this of the open and closed an is the of the The on of receptor conformation binding more this study on the conformation of the the conformation of the ligand bound to the receptor is the a of PTH(1–34) an conformation S. Scholar). studies of PTH and PTHrP with an in the and C-terminal by a region of 1998; Scholar, S. S. Scholar). bound to the receptor studies suggest an ligand conformation S. whereas studies suggest between the and C-terminal ligand T.J. Wilson Nussbaum Jr., J.T. Kronenberg Scholar). In this a of the binding of PTH(3–34) and is to with the conformation binds with affinity to the PTH1 receptor than PTH(3–34). the effect of PTH(1–14) binding on the affinity of PTH(3–34) that of the binding of PTH(3–34) is by receptor the portion of the a the of affinity of is to by the of a interaction of this region of the ligand with the juxtamembrane region of the The effect be for by the of the which in the of affinity of the ligand region with the for the have the two-site model to take into account conformations of the PTH1 the R and RG states. ligand interaction with the J domain of the PTH1 receptor the RG state the R state suggesting that the J interaction interaction and Ligand binding to the N domain is insensitive to R-G Finally, the increase of agonist affinity for the RG state a of the receptor the of the binding mechanism for these be to an of the signal mechanism of of this receptor Previous studies and ligands have a two-site of ligand by the PTH1 The C-terminal portion of the of PTH PTHrP interacts with the N-terminal receptor region (N interaction), and the N-terminal portion of the ligand binds to the juxtamembrane domain of the receptor (J H. Gardella T.J. Physiol. Scholar, Gardella T.J. Jr., J.T. Kronenberg H. Scholar). In this study the effect of receptor interaction on the mechanism of ligand binding to the PTH1 The of this study interaction for the receptor is described by the two-site for this binding was by the of between the binding of PTH(1–14) and PTH(3–34) to the receptor with the of simultaneous receptor binding of these ligands bind with higher affinity to the RG state than to the uncoupled whereas ligands bind with affinity to these states. The J interaction is by R-G coupling, whereas the N interaction is not by R-G more closed receptor conformation is suggested for the PTH1 receptor to in the model in The of between the binding of PTH(1–14) and PTH(3–34) the two-site model for the PTH1 The of simultaneous binding of PTH(1–14) and PTH(3–34) is with and of sites that with the and of the Gardella T.J. Jr., J.T. Kronenberg H. Scholar). The into the mechanism of PTH(3–34) PTH(1–14), to of PTH(3–34) with the juxtamembrane domain Jr., J.T. Gardella T.J. effect on the receptor binding affinity of 125I-PTH(3–34) increase of at the G-protein-uncoupled that PTH(3–34) not with the juxtamembrane domain, that the binding for PTH(3–34) is by the N The effect of on ligand binding to the receptor was by ligand affinity for the uncoupled receptor with the affinity for the RG The was measured by displacement of 125I-PTH(3–34) binding in the of 30 μm the a new radioligand that selectively the RG an of the bone T. Scholar). The R/RG of this radioligand was by the of binding by which the RG Annu. Rev. and by the increase of binding by of the PTH1 receptor with a that the RG state agonist ligands bound with higher affinity to the RG than to the uncoupled whereas ligands bound with affinity to these the of studies of the PTH1 receptor G. Endocrinology. and in with a of studies of S. T. Scholar). that G-protein by the RG state. of ligand binding to R and RG for a of to of In this study of the of ligand affinity for R and RG and the of 125I-[MAP]PTHrP(1–36) binding with affinity by the ligand The binding in this study be for by the of the model, was between these J.M. Scholar). of the receptor the and of the receptor in the model S. T. of the G-protein in with the to the the role of the G-protein in agonist binding to the PTH1 receptor to be the not agonist binding is required with the for the of the affinity agonist binding state Scholar, H. Scholar). the of agonist ligands was increased by R-G the to which the the two-site binding mechanism N were by R-G of the N-terminal region of the ligand the binding of the N-terminal residues PTH(1–34) in the of of of with the to a role for residue 5 in the of binding suggest that the affinity of ligand interaction with the J domain is increased by RG a by the of binding of PTH(1–14) to the RG state. In ligand binding to the N domain is not to RG coupling; PTH(3–34) bound with affinity to the R and RG of the R-G interaction ligand interaction with the J domain and not ligand binding to the N domain increase of ligand affinity for RG compared with R receptor conformations at the G-protein-coupled and receptor states. At the uncoupled the binding suggest than the conformation is open to the simultaneous binding of PTH(1–14) and PTH(3–34). the RG state, observations with the of a more closed receptor binding is suggesting that the ligand is the G. Scholar). PTH(1–14) a of the binding affinity of PTH(3–34) at the RG state, suggesting that simultaneous binding of ligand better binding of the PTH(3–34) of PTH(1–14)-stimulated cAMP accumulation. a of the PTH1 receptor to an conformation that to PTH(3–34) and PTH(1–14) bound to the The open and closed in this of the open and closed an is the of the The on of receptor conformation binding more this study on the conformation of the the conformation of the ligand bound to the receptor is the a of PTH(1–34) an conformation S. Scholar). studies of PTH and PTHrP with an in the and C-terminal by a region of 1998; Scholar, S. S. Scholar). bound to the receptor studies suggest an ligand conformation S. whereas studies suggest between the and C-terminal ligand T.J. Wilson Nussbaum Jr., J.T. Kronenberg Scholar). In this a of the binding of PTH(3–34) and is to with the conformation binds with affinity to the PTH1 receptor than PTH(3–34). the effect of PTH(1–14) binding on the affinity of PTH(3–34) that of the binding of PTH(3–34) is by receptor the portion of the a the of affinity of is to by the of a interaction of this region of the ligand with the juxtamembrane region of the The effect be for by the of the which in the of affinity of the ligand region with the In for the have the two-site model to take into account conformations of the PTH1 the R and RG states. ligand interaction with the J domain of the PTH1 receptor the RG state the R state suggesting that the J interaction interaction and Ligand binding to the N domain is insensitive to R-G Finally, the increase of agonist affinity for the RG state a of the receptor the of the binding mechanism for these be to an of the signal mechanism of of this receptor for and for of the