Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation

Abstract

The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an alpha-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

FIGURE 1.

Structure of the PTH1R ECD in the absence of ligand and comparison with PTH- and PTHrP-bound ECD structures. A, two views of the “ligand-free” PTH1R ECD dimer are shown. Subunit A of the dimer is colored orange, and subunit B is colored cyan. The four consensus N-linked glycosylation sites are shown in stick representation and colored yellow. The red lines represent the disordered loop (residues 59–105) that connect α1 and β1. MBP is not shown for clarity. B, omit electron density maps were calculated with the C-terminal α-helix (α2) of the ECD removed from the model. The 2F_o − F_c map is shown as a blue mesh contoured at 1 σ, and the F_o − F_c map is shown as a green mesh contoured at 3 σ. Subunit B α2 is shown in stick representation. The last two residues modeled are the first two histidine residues of the His₆ tag, denoted H188* and H189*. C, shown is the molecular surface of subunit A showing the hydrophobic groove that forms the base of the peptide binding site. Carbon atoms are colored gray, oxygen atoms are red, and nitrogen atoms are blue. Subunit B α2 is shown as a cyan coil, and the remainder of subunit B is shown as a molecular surface with carbon atoms colored cyan. D, shown is a detailed view of the interaction between subunit B α2 and the subunit A ECD. α2 is shown as a cyan coil with selected side chains shown as sticks. Hydrogen bonds are depicted as red dashes. E, shown is structural alignment of the subunit A ECD-subunit B α2 complex from the ligand-free dimer structure and the ECD-PTH (PDB code 3C4M) and ECD-PTHrP (PDB code 3H3G) complexes. Subunit B α2 of the ECD dimer is shown as a cyan coil, PTH as a yellow coil, and PTHrP as a magenta coil. The subunit A ECD of the dimer is shown in orange, the PTH-bound ECD is green, and the PTHrP-bound ECD is slate blue. Selected side chains are shown in stick representation. F, amino acid sequence alignment of residues 172–191 of human PTH1R and the 1–34 fragments of human PTH and PTHrP is shown.

FIGURE 2.

BRET analysis of PTH1R oligomerization in COS-1 cells. A and B, static BRET ratios obtained from cells co-expressing the indicated Rlu- and YFP- tagged PTH receptor constructs. The shaded area represents the nonspecific BRET signal (∼0.03) obtained between Rlu-tagged PTH1 receptor and YFP-tagged CCK₂ receptor (CCK₂R-YFP). Levels of significance: **, p < 0.01 from wild-type receptor; and data are the means ± S.E. of 4–5 experiments performed in duplicate. C and D, shown are saturation BRET curves for the indicated receptors. E, BRET signals derived from the effect of PTH ligand on the PTH1 receptor homodimer are shown. Data are the means ± S.E. of 4–5 experiments performed in duplicate.

FIGURE 3.

PTH binding and cAMP signaling ability of wild-type and mutant PTH receptors with alterations in the ECD α2 helix or ECD peptide binding site. A, shown is a whole cell radioligand binding assay assessing the ability of COS-1 cells expressing the indicated constructs to bind ¹²⁵I-PTH-(1–34). The data represent the mean ± S.E. of triplicate samples. B, shown is displacement binding with cold PTH-(1–34) competitor for the indicated constructs. The data represent the mean ± S.E. of triplicate samples. C and D, shown is a dose-response assay for cAMP accumulation. COS cells expressing the indicated constructs were stimulated with PTH for 30 min at 37 °C. The data represent the mean ± S.E. of duplicate samples. The EC₅₀ values for WT, R179A/V183A/L187A, R179A/V183A, and V183A receptors in C were 60, 144, 109, and 72 pm, respectively. The EC₅₀ values for WT, R179A, L187A, I135K, and D137A receptors in D were 86 pm, 73 pm, 482 pm, 31 nm, and 3 nm, respectively.

FIGURE 4.

Oligomerization and cAMP signaling ability of mutant PTH1 receptors with alterations in the ECD α2 helix designed to stabilize the dimer by more closely mimicking PTH/PTHrP binding. A, static BRET ratios for the indicated receptors expressed in COS-1 cells are shown. B, dose-response assay for cAMP accumulation in COS cells expressing the indicated receptors is shown. The data represent the mean ± S.E. of duplicate samples. The EC₅₀ values for WT, E182F/R186L, E182W/R186L, E182F/V183L/R186L, and E182W/V183L/R186L receptors were 36, 64, 86, 66, and 98 pm, respectively.

FIGURE 5.

Morphological FRET analysis of PTH1R oligomerization. Shown are representative corrected microscopic images of fixed COS cells expressing CFP- and YFP-tagged constructs as indicated. The images represent background-subtracted CFP, background-subtracted YFP, and corrected FRET signals. The scale bar represents 25 μm.

FIGURE 6.

Model for the intact, ligand-free PTH1R dimer and PTH disruption of the dimer. A, the model depicts the only orientation of the ECD dimer with respect to the membrane that is possible given the location of the ECD C termini of the two subunits. As modeled, an ∼90° turn between the ECD α2 helix and the first transmembrane helix (TM1) would be required. The four consensus sites of N-linked glycosylation are shown in yellow. The membrane-embedded 7-TM domains are shown as squares that approximate the size of a 7-TM helical bundle relative to the ECDs. The specific arrangement of the individual 7-TM domains with respect to each other and the attached ECD is not clear, but the 7-TM domains apparently do not significantly contribute to dimerization. PTH is shown as a yellow helix modeled by positioning the crystal structure of PTH-(1–34) (PDB code 1ET1) into the peptide binding site of the ECD. B, view of the ECD with docked PTH-(1–34) showing a surface representation of PTH to highlight the hydrophobic face of the 1–14 segment, which is on the opposite side of the helix as the hydrophobic face of the 15–34 segment that contacts the ECD peptide binding groove. Carbon atoms are colored yellow, sulfur atoms are orange, nitrogen atoms are blue, and oxygen atoms are red. As depicted in our model, the hydrophobic face of the 1–14 segment would face toward the membrane and contact the helical bundle of the 7-TM domain.