Conserved class B GPCR activation by a biased intracellular agonist.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

Constitutively active PTH/PTHrP receptor specifically expressed in osteoblasts enhances bone formation induced by bone marrow ablation.

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1-constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady-state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild-type mice. Whereas de novo bone formation was restricted to the ablated area in wild-type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase-positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt-signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation-induced recruitment, proliferation, and differentiation of osteoprogenitors.

Functional type I PTH/PTHrP receptor in freshly isolated newborn rat keratinocytes: identification by RT-PCR and immunohistochemistry.

The presence of identical or distinct type I parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptors in keratinocytes is still a matter of debate. We studied the expression and functionality of PTHrP receptors in freshly isolated keratinocytes from newborn rat skin. Four overlapping primers, amplifying different regions in the rat PTH receptor, were used for reverse transcriptase-polymerase chain reaction (RT-PCR). The first region corresponded to the N-terminal extracellular region and the first transmembrane domain (S/M1), the second region amplified the connecting intracellular and extracellular loops transmembrane domain (E2/M5), the third spanned the range from the transmembrane to the intracellular domain (M4/T), and the fourth region amplified the C-terminal tail (M6/7/T). The PCR products from the keratinocyte RNA were identical to those from kidney RNA of the same rats. The cloned four transcripts showed 100% of homologies with the cDNA sequence from bone ROS cells. Keratinocytes, freshly isolated or present in situ in the epidermis, recognized an anti-PTH receptor antibody (PTH-II) directed against the receptor extracellular domain. Western blotting showed the same protein patterns in keratinocytes, kidney, and ROS cell extracts. Low doses of PTHrP(1-34) (10(-12)-10(-9) M) increased the cell number studied by [3H]thymidine incorporation and DNA content. Treatment with the PTH/PTHrP receptor antagonist [Asn10, Leu11, D Trp12] PTHrP(7-34) or two different PTH receptor antibodies inhibited the increase in cell proliferation induced by PTHrP(1-34). All these findings indicate that newborn rat epidermis and keratinocytes express functional PTHrP receptors, which are identical to type I PTH/PTHrP receptor and are recognized by PTHrP(1-34).