Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor

Abstract

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G_s and increased activation of G_q compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

Keywords: GPCRs; biased signaling; ectodomain cleavage; matrix metalloproteinase; parathyroid hormone 1 receptor.

Figure 1

Topology of PTH1R. Structure of the human PTH1R (transmembrane domain, grey; ECD, teal) in complex with a PTH analog (orange) (PDB ID: 6FJ3). Unstructured residues 61-105 of ECD loop 1 are depicted as a dashed line. The receptor N-terminus residue (V³¹, as resolved in the crystal structure), residues embracing ECD loop 1, and transmembrane helices (TM1-7) are indicated.

Figure 2

Mapping of the protease cleavage site of PTH1R ECD by alanine scan. (A) Amino acid sequence of the extracellular domain of the human PTH1R. Stretches of 8 amino acids that were replaced by alanine residues are indicated by horizontal bars. Exon E2 is marked above the sequence. (B) CHO cells stably expressing PTHR variants were treated with 100 nM PTH(1-34) for 30 min or left untreated. Subsequently, cells were fixed, permeabilized, and stained with rabbit anti-PTH1R antibody followed by Cy2-labeled anti-rabbit antibody. PTHR was visualized by confocal microscopy. (C) CHO cells stably expressing PTHR variants were lysed, and PTH1R was monitored by reducing SDS-PAGE and Western blotting. Cells were treated with 100 nM PTH(1-34) for 12 h prior to cell lysis where indicated. Arrowheads depict the cleaved (MW ~80 kDa) and the uncleaved (MW ~90 kDa) PTH1R band. n.s., not stimulated

Figure 3

Mapping of the protease cleavage site of PTH1R ECD by N-terminal sequencing. (A) Human PTH1R with C-terminal Strep-Tag II was purified from stably expressing CHO cells by two-step affinity purification. A fraction of the purified receptor was subjected to Western blot and probed with anti-PTH1R antibodies (left panel). The remaining purified receptor protein (~50 µg) was transferred on PVDF membranes and stained with Coomassie blue R250. The band corresponding to PTH1R was cut out and subjected to microsequencing (right panel, dashed box). (B) Sequence of exon E2 (amino acids 61-105). Sequences obtained from microsequencing are shaded gray. The position of the N-terminal amino acid is marked by an arrow. Residues 84-86 (gray diagonal stripes) were not resolved in the Edman degradation.

Figure 4

Protease cleavage changes the signaling specificity of PTH1R from G_q and G_s. (A) Flp-In CHO cells stably expressing PTH1R, PTH1R^56-63A or PTH1R^72-79A were stimulated for 20 min with PTH(1-34) at the indicated concentrations, and cAMP levels were quantified with a radioimmunoassay. The means ± S.E.M. of five independent experiments are shown. (B) Flp-In CHO cells stably expressing PTH1R, PTH1R^56-63A or PTH1R^72-79A were incubated with [myo-2-³H(N)]inositol and 0.2% fetal calf serum for 16 h. Cells were stimulated for 60 min with the indicated concentrations of PTH, and [³H]IP₃ levels were quantified in a scintillation counter after chromatographic separation. Data represent the means ± S.E.M. of five individual experiments.

Figure 5

Interface between PTH and α1-helix of PTH1R ECD. Crystal structure of the PTH1R ECD (teal) in complex with PTH (orange) (PDB ID: 6FJ3). Residues forming the interface are shown as stick, and contacts are indicated as blue dashed lines. The unstructured loop 1 of the ECD is shown as black dashed line.