Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the PTH/PTH-related peptide (PTHrP)receptor on hormone binding and receptor activation

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II fam ily of G protein-coupled receptors. To delineate the sites in the PTH1r's N -terminal region, and the carboxy-core domain (transmembrane segments + ext racellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK- 293 cells. The wild type and chimeric receptors were analyzed for cell surf ace expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chi meric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were re placed either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in respons e to PTH. This data indicate that receptor activation is dissociable from h igh affinity hormone binding in the PTH1r, and that the N-terminal region m ight play a critical role in the activation process. Further segment replac ements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. T he data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residu es 1-62) (site 1) that participates in high-affinity PTH binding; and a sec ond site of lower affinity constituted by amino acid residues scattered thr oughout the carboxy-terminal half (residues 105-186) (site 2). In the absen ce of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss o f high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first-and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affini ty and did not signal in response to PTH. Further substitution of helix 5 o f PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signali ng response. Additional substitutions of PTH1r's helices in various combina tions emphasize the fundamental role of helix 3 and helix 6 on the activati on process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute dif ferentially to PTH binding affinity and signal transduction mechanism and h ighlight the role of the N-terminal domain and helix 3 and helix 6 on recep tor activation.