PTH and PTH antagonist induce different conformational changes in the PTHR1 receptor

Abstract

Interaction of ligands with their specific receptors is accompanied by conformational shifts culminating in receptor activation and expression of hormonal activity. Using an engineered disulfide bond formation strategy, we characterized the relative conformational changes taking place within the PTH type 1 receptor (PTHR1) at the interface of transmembrane (TM)5 and TM6 on binding the PTH agonist, PTH(1-34), compared with the antagonist PTH(7-34). Cysteines were singly incorporated into a portion of the extracellular-facing region of TM5 (365-370), while simultaneously a second cysteine was introduced at position 420, 423, or 425 at the extracellular end of TM6, leading to a total of 18 double cysteine-containing PTHR1 mutants. All mutants, except P366C/V423C and P366C/M425C, were expressed in the cell membrane preparations. In the presence of agonist, H420C and M425C in TM6 formed disulfide bonds with all and with most, respectively, of the substituted cysteines incorporated in TM5. In contrast to the conformational shift induced (or stabilized) by agonist in activating the receptor, antagonist binding produced no detectable change from the basal (inactive) conformation of PTHR1. Our studies provide physicochemical evidence that the extracellular-facing ligand binding regions of receptor, TM5 and TM6, are dynamic and move relative to each other on ligand binding. The distinct differences in receptor conformation induced (or stabilized) by agonist PTH(1-34) compared with antagonist PTH(7-34) begin to provide insight into the early events in and mechanism of PTHR1 activation.

FIG. 1

Schematic of PTHR1 template. Shown are amino acid residues V365, P366, I367, L368, A369, and S370 in TM5 and H420, V423, and M425 in TM6 of PTHR1 that were substituted with Cys in this study. Also shown is the location of two FXa cleavage sites in IC3. For detection by Western blot, a c-myc tag was inserted between residues 572 and 573 of C-ICD. The endogenous cysteines are shown as open circles (○).

FIG. 2

Rationale for experimental approach for detecting disulfide bond formation or disruption between engineered cysteines in the modified PTHR1. PTHR1 constructs containing introduced cysteines were incubated with PTH agonist or antagonist. If a disulfide bond forms (right), digestion with FXa will yield a band of size equal to undigested receptor. The receptor is held together by the disulfide bridge despite cleavage by FXa. However, if a disulfide bond is not formed (left), a low molecular weight (∼23 kDa) C-terminal fragment (indicated by arrow) containing the c-myc epitope will be detected by Western blot with anti-c-myc antibody.

FIG. 3

Expression of PTHR1s in cell membrane preparations. COS-7 cells transiently transfected with wtPTHR1, XM-PTHR1, and various double cysteine-containing PTHR1 mutants were used to prepare membrane preparations. Receptor expression was detected by Western blot using anti-c-myc antibody as probe. The lanes are (1) untransfected control, (2) wtPTHR1, (3) XM-PTHR1, (4)V365C/M425C, (5) P366C/M425C, (6) I367C/M425C, (7) L368C/M425C, (8) A369C/M425C, (9) S370C/M425C, (10) V365C/V423C, (11) P366C/ V423C, (12) I367C/V423C, (13) L368C/ V423C, (14) A369C/V423C, (15) S370C/V423C, (16) V365C/H420C, (17) P366C/H420C, (18) I367C/H420C, (19) L368C/H420C, (20) A369C/H420C, and (21) S370C/ H420C.

FIG. 4

Detection of relative internal conformational shifts of TM5 and TM6 when PTH agonist or antagonist binds to receptor. Western blots of TM5/TM6 double cysteine-containing mutant PTHR1s probed with anti-c-myc antibody are shown. COS-7 cells transfected with XM-PTHR1 and the double cysteine-substituted XM-PTHR1 constructs shown were used to make membrane preparations. One hundred micrograms of these membrane preparations was treated with FXa after treatment with oxidizing agent alone or with oxidizing agent plus PTH(1-34) or PTH(7-34) as indicated. (Left) Samples run under nonreducing conditions. (Right) Same sample sets run under reducing conditions. Only the low MW band of interest (∼23 kDa) is detected. A decrease in intensity indicates enhanced disulfide bond formation across TM5/TM6. Conversely, increased intensity indicates diminished disulfide bond formation (or disruption of formation).

FIG. 5

Evidence for ligand dose-dependent formation of disulfide bonds. Western blots of mutant receptor-transfected membrane preparations are shown. Double cysteine-containing XM-PTHR1 mutants were incubated with different concentrations of agonist PTH(1-34) or antagonist PTH(7-34). After SDS-PAGE of samples processed under nonreducing and reducing conditions, anti-c-myc antibody was used to detect the low MW (∼23 kDa) band.

FIG. 6

Illustration of ligand-bound PTHR1. A molecular model of the PTH(1-34)/PTHR1 complex, as viewed from the extracellular surface, looking into the cell, as developed from structural, photoaffinity, and disulfide binding studies. The receptor is blue; PTH(1-34) is red, and the residues of TM5 and TM6 examined here are denoted.