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

Abstract

As the oxytocin receptor plays a key role in parturition and lactation, there is considerable interest in defining its structure/functional relationships. We previously showed that the rat oxytocin receptor transfected into Chinese hamster ovary cells was coupled to both G q/11 and G i/o , and that oxytocin stimulated ERK-2 phosphorylation and prostaglandin E 2 synthesis via protein kinase C activity. In this study, we show that deletion of 51 amino acid residues from the carboxyl terminus resulted in reduced affinity for oxytocin and a corresponding rightward shift in the dose-response curve for oxytocin-stimulated [Ca 2؉ ] i . However, oxytocin-stimulated ERK-2 phosphorylation and prostaglandin E 2 synthesis did not occur in cells expressing the truncated receptor. Oxytocin also failed to increase phospholipase A activity or activate protein kinase C, indicating that the mutant receptor is uncoupled from G q -mediated pathways. The ⌬51 receptor is coupled to G i , as oxytocin-stimulated Ca 2؉ transients were inhibited by pertussis toxin, and a G␤␥ sequestrant. Preincubation of ⌬51 cells with the tyrosine kinase inhibitor, genistein, also blocked the oxytocin effect. A ⌬39 mutant had all the activities of the wild type oxytocin receptor. These results show that the portion between 39 and 51 residues from the COOH terminus of the rat oxytocin receptor is required for interaction with G q/11 , but not G i/o . Furthermore, an increase in intracellular calcium was generated via a G i ␤␥-tyrosine kinase pathway from intracellular stores that are distinct from G q -mediated inositol trisphosphate-regulated stores. Oxytocin (OT)1 is a nine-amino acid peptide that stimulates uterine smooth muscle and mammary myoepithelial cell contraction, and prostaglandin production by uterine endometrial and amnion cells. Nucleic acid sequencing of cDNA clones of the oxytocin receptor (OTR) indicated that it is a member of the G protein-coupled receptor (GPCR) superfamily (1). As OT plays a pivotal role in parturition and lactation, there is considerable interest in defining the structure of the OTR. It has been shown for a number of GPCRs that several regions in the cytoplasmic domains contribute directly or indirectly to G protein coupling (see Ref. 2 for a review). 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). The COOH terminus of the human parathyroid h...

Localization of the human oxytocin receptor in caveolin-1 enriched domains turns the receptor-mediated inhibition of cell growth into a proliferative response

“…It is interesting to note that this PAR2 mutant showed the same defective internalization and desensitization as the OTR-GFP-cav2 mutant described here. Since human OTR activates the p42/44 and p38 MAPK pathways via both sensitive and insensitive PTX signalling pathways (Hoare et al, 1999;Ohmichi et al, 1995;Strakova et al, 1998), it can be hypothesized that OTR localized in caveolar domains activate the MAP kinase in a Ras-dependent pathway leading to the activation and translocation of ERK1/ERK2 and cell proliferation, whereas the inhibition of cell growth may be associated with PLC-and PCK-dependent and Ras-independent activation. Experiments using inhibitors of the dierent signalling pathways potentially involved in MAPK activation by OTR-GFP-cav2 or WT OTR-GFP are currently under way in order to test this hypothesis.…”

“…It has been shown that OTRs couple to Ga q/11 alone, to Ga i and Ga q/11 , or to Ga i and Ga s in myometrial cells (Hoare et al, 1999;Strakova and Solo, 1997), and that the inhibitory eects on the proliferation of breast cancer and glial cells are associated with an increase in intracellular cAMP levels (Cassoni et al, 1997(Cassoni et al, , 1998. Furthermore, since OT promotes the synthesis of prostaglandins in a number of cell systems, some OTmediated eects on cell proliferation may be indirectly mediated by an autocrine/paracrine loop involving the activation of Ga s -coupled prostaglandin receptors, thus leading to increased cAMP production and the inhibition of proliferation.…”

Multiple Roles for the C-terminal Tail of the Chemokine Scavenger D6

“…The recent crystal structures of the human ␤ 2 -adrenergic receptor have an 8th helix underlying the membrane perpendicular to the transmembrane helices which is stabilized by a hydrophobic surface (40 -43). In fact, an 8th helix is thought to be present in most, if not all, rhodopsin-like 7TMRs and play a role in signaling by directly interacting with heterotrimeric G-protein subunits (43)(44)(45)(46)(47)(48)(49), although roles in ligand binding have also been reported (50). The 8th helix of D6 (Fig.…”

Helix 8 of the Viral Chemokine Receptor ORF74 Directs Chemokine Binding

“…Although for some GPCRs, Hx8 appears dispensable for signaling (26), the importance of Hx8 in G-protein interaction has been shown, e.g. for the oxytocin receptor (27), bradykinin receptors (28), the angiotensin II receptor type 1A (29), the leukotriene B4 receptor (30), and the ␤ 1 -adrenergic receptor (31). Moreover, the N-terminal portion of Hx8 of the protease-activated receptor PAR1 was found to be involved in coupling to G␣ q , through a network of H-bond and ionic interactions, connecting Hx8 to the conserved NPXXYX 5,6 F motif on TM7 and also to the adjacent intracellular loop 1 (32).…”