A C‐terminally truncated human parathyroid hormone receptor is functional and activates multiple G proteins

Abstract

We have investigated the role of the C-terminal cytoplasmic domain of the human PTH receptor in effector coupling. Following transient expression in COS-1 cells, coupling to both AC and PI-PLC was observed with the full-length receptor. Progressive C-terminal truncations did not dissociate activation of the two signalling systems. In stably transfected 293 cells, however, the full-length receptor as well as the majority of truncated constructs stimulated AC exclusively but failed to activate PI-PLC. Activation of both signalling systems was again observed following stable expression of a severely truncated receptor (R483) in 293 cells. In this case, pertussis toxin was also found to potentiate the cAMP response to hPTH-(1-38) significantly, indicating functional coupling of R483 to Gi proteins. Our results suggest that a core region of the human PTH receptor (first, second, third intracellular loop) can interact promiscuously with different G proteins and that the C-terminus of the full-length receptor directs the receptor towards an interaction with G~.Key words." Parathyroid hormone; Receptor; G protein; Adenylyl cyclase; Phosphoinositide 1, IntroductionPTH is a major regulator of bone and mineral metabolism. In its target cells, which are primarily osteoblasts and kidney proximal tubule cells, the hormone elicits a multitude of responses which are not readily explained solely on the basis of its capacity to stimulate AC (see [1,2] for review). Receptors for PTH have been cloned from opossum, rat, and human cDNA libraries [3][4][5][6]. These receptors bind PTH, PTH-related peptide, and N-terminal fragments of these hormones with nanomolar affinities. Only one PTH receptor gene exists in the mouse, rat, and human genome [7] and so far, there is no evidence lbr receptor subtypes mediating different physiological effects [5].Importantly, the rat and human receptor activate both cAMP and IP production following transient expression in COS cells [4,6]. Stimulation of both signal transduction systems has also been observed with the structurally related calcitonin receptor [8]. Studies presented in abstract form [9] concerning the full-length and a truncated opossum PTH receptor (OK-H, see Fig. 1) had suggested that regions within the C-terminus of the PTH receptor play an important role in the activation of PI turnover, whereas they are dispensable for the coupling to AC.Given these results, we set out to study the coupling specificity of the full-length human PTH receptor, which we had recently cloned from a kidney cDNA library [6], as well as a number of receptor constructs truncated in the C-terminal intracellular tail region. Coupling to AC and PI-PLC was inves-*Corresponding author. Fax: (41) (61) 324 4774.Abbreviations: AC, adenylyl cyclase; Gs and G~, stimulatory and inhibitory G protein of adenytyl cyclase, respectively; Gq, stimulatory G protein of PI-PLC; IP, inositol phosphate; PI, phosphoinositide; PI-PLC, phosphoinositide-specific phospholipase C; PTH, parathyroid hormone; hPTH, human parathyroi...

Characterization of parathyroid hormone/parathyroid hormone-related protein receptor and signaling in hypercalcemic Walker 256 tumor cells

“…On the other hand, we demonstrated the existence of a PTHrP receptor coupling to G q/11 , which was detected in W256 membranes by Western blot analysis, in these cells. Previous studies with the full-length and truncated forms of the PTHR stably transfected in 293 cells showed that a region of the C-terminus beyond residue 483 of this receptor modulates G s activation (Schneider et al 1994). Moreover, using COS-7 cells transiently expressing various PTHR constructs, the C-terminal region of this receptor has been shown to lower the apparent receptor affinity, and to decrease the efficacy of cAMP responsiveness without altering phospholipase C activation (Iida-Klein et al 1995).…”

The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to Gq, but Not Gi

“…The COOH-terminal domain of some GPCRs plays an important role in G protein isotype selectivity (11,12). At least four isoforms of the prostaglandin EP3 receptor, differing only at their COOH-terminal tails (produced by alternative splicing), couple to different G proteins to activate different second messenger systems (13,14).…”

“…At least four isoforms of the prostaglandin EP3 receptor, differing only at their COOH-terminal tails (produced by alternative splicing), couple to different G proteins to activate different second messenger systems (13,14). The COOH terminus of the human parathyroid hormone receptor directs the receptor toward an interaction with G s , whereas a core region composed of the first, second, and third intracellular loops can interact promiscuously with different G proteins (11). A truncated human AT1 receptor mutant lacking the carboxyl-terminal 50 residues is deficient in coupling to G i , but it retains full ability to bind to G q (12).…”

“…The juxtamembrane portions of cytoplasmic loop 3 and cytoplasmic loop 2 of several family members have been implicated in receptor-G protein interactions. In addition, the COOH-terminal region of adrenergic receptors (3, 4) and other receptor types (5-7) is also required for G protein interactions, but this domain does not appear to be important for receptor function of all GPCR family members (8 -10).The COOH-terminal domain of some GPCRs plays an important role in G protein isotype selectivity (11,12). At least four isoforms of the prostaglandin EP3 receptor, differing only at their COOH-terminal tails (produced by alternative splicing), couple to different G proteins to activate different second messenger systems (13,14).…”

SH3 Binding Domains in the Dopamine D4 Receptor

“…In some cases, as seen with the P-adrenergic receptor, deletion of part of the carboxyl-tenninal tail leads to improved G proteincoupling efficiency (Parker and Ross,199 1 ), increased basal activity (Parker and Ross, 199 1 ), or increased prorniscuity for different G protein-coupling (Schneider et al, 1994). Complete removal of the carboxyl-terminal tail f?om GPCRs results in the inactivation of the receptor (Sano et al, 1997;Segaloff and Ascoli, 1993).…”