Increase In Inositol Phosphate Production Research Articles

Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist.

As a biased agonist, peptide angiotensin II (Ang II) type 1 (AT1) receptor ligand antagonizes Ang II-stimulated G protein signaling but stimulates several kinase pathways. Here, we developed a non-peptide AT1 receptor compound as a biased ligand. We synthesized three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) as candidates of biased ligands. Extracellular signal-regulated kinase (ERK) 1/2 activation and inositol phosphate (IP) production were measured using a cell system that overexpressed AT1 receptors (wild-type, L112A, Q257A, Y292A, and N295A receptors). We also examined the modes of receptor-ligand binding using a competition binding study. The Kd values of R239470, R781253, and R794847 for the AT1 wild-type receptor were 0.8, 21, and 48 nM, respectively, as assessed in a competition binding study. Those of R239470, R781253, and R794847 for the L112A receptor were 37, 23, and 31 nM, respectively. R781253 and R794847 decreased and increased IP production, respectively, whereas R239470 did not change IP production. R781253 and R794847, but not R239470, activated ERK1/2. In conclusion, R239470, R781253, and R794847 act as a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, R781253 and R794847, but not R239470, are agonists toward ERK1/2 activation. Thus, we developed a non-peptide AT1 receptor compound as a biased ligand.

PS-BPB08-2: DEVELOPMENT OF NON-PEPTIDE TYPE OF ANGIOTENSIN II TYPE 1 RECEPTOR LIGAND AS A BIASED AGONIST

Objective: Peptide type of angiotensin II (Ang II) type 1 (AT1) receptor ligand as a biased agonist antagonized Ang II-stimulated G protein signaling, but stimulated several kinase pathways. Here, we developed non-peptide type of AT1 receptor compound as a biased ligand. Design and method: We synthesized three non-peptide type of AT1 receptor ligands (Compound A, B and C) as candidates of biased ligands. These compounds were provided by Daiichi Sankyo Co. LTD. In vitro studies, inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) 1/2 activation were measured using AT1 receptor overexpressing cell system. We mutated the AT1 receptor (L112A, Q257A, Y292A and N295A receptors) and examined binding modes of receptor-ligand by competition binding study, and subsequently analyzed whether these ligands would induce IP production and ERK1/2 activation. Results: Kd of Compound A, B and C to AT1 wild-type receptor by competition binding study were 0.8, 21 and 48 nM, respectively. Kd of Compound A, B and C to L112A receptor were 37, 23 and 31 nM, respectively. Compound B and C decreased and increased IP production, respectively, whereas Compound A did not change IP production. Compound B and C, but not Compound A, activated ERK1/2. L112A had a key role of IP production. Conclusions: Compound A, B and C were a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, Compound B and C, but not Compound A, were agonists to ERK1/2 activation. Thus, we developed non-peptide type of AT1 receptor compound as a biased ligand.

Frequent and Persistent PLCG1 Mutations in Sézary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling

Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.

A Second Disulfide Bridge from the N-Terminal Domain to Extracellular Loop 2 Dampens Receptor Activity in GPR39

A highly conserved feature across all families of 7TM receptors is a disulfide bridge between a Cys residue located at the extracellular end of transmembrane segment III (TM-III) and one in extracellular loop 2 (ECL-2). The zinc sensor GPR39 contains four Cys residues in the extracellular domains. By using mutagenesis, treatment with the reducing agent TCEP, and a labeling procedure for free sulfhydryl groups, we identify the pairing of these Cys residues in two disulfide bridges: the prototypical bridge between Cys (108) in TM-III and Cys (210) in ECL-2 and a second disulfide bridge connecting Cys (11) in the N-terminal domain with Cys (191) in ECL-2. Disruption of the conserved disulfide bond by mutagenesis greatly reduced the level of cell surface expression and eliminated agonist-induced increases in inositol phosphate production but surprisingly enhanced constitutive signaling. Disruption of the nonconserved disulfide bridge by mutagenesis led to an increase in the Zn (2+) potency. This phenotype, with an approximate 10-fold increase in agonist potency and a slight increase in E max, was mimicked by treatment of the wild-type receptor with TCEP at low concentrations, which had no effect on the receptor already lacking the second disulfide bridge and already displaying a high Zn (2+) potency. We conclude that the second disulfide bridge, which according to the beta2-adrenergic structure will form a covalent link across the entrance to the main ligand binding pocket, serves to dampen GPR39 activation. We suggest that formation of extra disulfide bridges may be an important general mechanism for regulating the activity of 7TM receptors.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Involvement of the Ser‐Glu‐Pro Motif in Ligand Species‐Dependent Desensitisation of the Rat Gonadotrophin‐Releasing Hormone Receptor

There are two forms of gonadotrophin-releasing hormone (GnRH), GnRH-I and GnRH-II, in the vertebrate brain. Both GnRH-I and GnRH-II are thought to interact with the type-I GnRH receptor (GnRHR). The present study attempted to demonstrate whether GnRH-I and GnRH-II induce differential desensitisation of GnRHR and to identify the motif involved. Time course inositol phosphate (IP) accumulation assay reveals that, in cells expressing the wild-type rat GnRHR, GnRH-I induced continuous increase in IP production, whereas GnRH-II-induced IP production rate at later time points (30-120 min after ligand treatment) became attenuated. However, in cells expressing the mutant receptor in which the Ser-Glu-Pro (SEP) motif in extracellular loop 3 was replaced by Pro-Glu-Val (PEV), IP accumulation rates at later time points were more decreased by GnRH-I than GnRH-II. Ca2+ responses to repetitive GnRH applications reveal that GnRH-II desensitised the wild-type receptor faster than GnRH-I, whereas the opposite situation was observed in the PEV mutant. In addition, cell surface loss of GFP-tagged wild-type receptor was more facilitated by GnRH-II than GnRH-I, whereas that of the GFP-tagged PEV mutant receptor was more enhanced by GnRH-I than GnRH-II. The present study indicates that the SEP motif is potentially responsible for ligand species-dependent receptor desensitisation. Together, these results suggest that GnRH-I and GnRH-II may have different effects on mammalian type-I GnRHR via modulation of desensitisation rates.

Gα12/13- and Rho-Dependent Activation of Phospholipase C-ϵ by Lysophosphatidic Acid and Thrombin Receptors

Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of Galpha(12/13) and Rho.

Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes

The extracellular Ca(2+)-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR(-/-)) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [(Exon5(-))CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR(-/-) mice express (Exon5(-))CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of (Exon5(-))CaR in growth plates from CaR(-/-) mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human (Exon5(-))CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human (Exon5(-))CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR(-/-) GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR(-/-) mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably (Exon5(-))CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR(-/-) mice, high extracellular [Ca(2+)] ([Ca(2+)](e)) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca(2+)](e) also promoted the differentiation of CaR(-/-) GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of (Exon5(-))CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca(2+)](e) in GPCs in CaR(-/-) mice.

Dual Role of Histamine in Modulation of Leydig Cell Steroidogenesis via HRH1 and HRH2 Receptor Subtypes1

Although several reports indicate effects of histamine (HA) on female reproductive functions, scant literature exists to suggest a physiological role of HA in the male gonad. In the present study, we report a dual concentration-dependent effect of HA on steroidogenesis in MA-10 murine Leydig cells and purified rat Leydig cells. Although 1 nM HA can stimulate steroid production and significantly increase the response to LH/hCG in these cells, 10 microM HA exerts an inhibitory effect. We also provide confirming evidence for the existence of functional HRH1 and HRH2 receptors in both experimental models. The use of HRH1 and HRH2 selective agonists and antagonists led us to suggest that HRH2 activation would be largely responsible for stimulation of steroidogenesis, while HRH1 activation is required for inhibition of steroid synthesis. Our results regarding signal transduction pathways associated with these receptors indicate the coupling of HRH2 to the adenylate cyclase system through direct interaction with a Gs protein. Moreover, we show HRH1 activation mediates increases in inositol phosphate production, possibly due to coupling of this receptor to Gq protein and phospholipase C activation. The data compiled in this report clearly indicate that HA can modulate Leydig cell steroidogenesis in the testis and suggest a possible new physiological site of action for HA. Given that many drugs binding to HRH1, HRH2, or both, are widely prescribed for the treatment of diverse HA-related pathologies, it seems necessary to increase the knowledge regarding histaminergic regulation of testicular functions, to avoid possible unexpected side effects of such substances in the testis.

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr 4+, Ga 3+, Al 3+, Fe 3+, Lu 3+, and Sc 3+, were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [ 3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [ 3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [ 3H]inositol phosphate production in cells preincubated with [ 3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.

Alpha 1-adrenoceptor effects mediated by protein kinase C alpha in human cultured prostatic stromal cells.

1 We have investigated the effects of alpha(1)-adrenoceptor stimulation upon contractility, Ca(2+) influx, inositol phosphate production, and protein kinase C (PKC) translocation in human cultured prostatic stromal cells (HCPSC). 2 The alpha(1)-adrenoceptor selective agonist phenylephrine elicited contractile responses of HCPSC, i.e. a maximal cell shortening of 45+/-6% of initial cell length, with an EC(50) of 1.6+/-0.1 microM. The alpha(1)-adrenoceptor selective antagonists prazosin (1 microM) and terazosin (1 microM) both blocked contractions to phenylephrine (10 microM). The L-type calcium channel blocker nifedipine (10 microM), and the PKC inhibitors Gö 6976 (1 microM) and bisindolylmaleimide (1 microM) also inhibited phenylephrine-induced contractions. 3 Phenylephrine caused a concentration dependent increase in inositol phosphate production (EC(50) 119+/-67 nM). This response was blocked by terazosin (1 microM). 4 Phenylephrine caused the translocation of the PKC alpha isoform, but not the beta, delta, gamma, epsilon or lambda isoforms, from the cytosolic to the particulate fraction of HCPSC, with an EC(50) of 5.7+/-0.5 microM. 5 In FURA-2AM (5 microM) loaded cells, phenylephrine elicited concentration dependent increases in [Ca(2+)](i), with an EC(50) of 3.9+/-0.4 microM. The response to phenylephrine (10 microM) was blocked by prazosin (1 microM), bisindolymaleimide (1 microM), and nifedipine (10 microM). 6 In conclusion, this study has shown that HCPSC express functional alpha(1)-adrenoceptors, and that the intracellular pathways responsible for contractility may be largely dependent upon protein kinase C activation and subsequent opening of L-type calcium channels.

The discovery of novel small molecule non-peptide gonadotropin releasing hormone (GnRH) receptor antagonists

A novel series of non-peptide derivatives 1, 14, and 15 that bind with high affinity to the human GnRH receptors is discussed. The discovery was made from screening our in-house libraries that contained the active structure 2 along with a trace amount of a second active structure 1 that was derived from an acid-induced rearrangement. From this structure type 1, a series of guanidine and non-guanidine containing analogues were prepared and tested as GnRH receptor antagonists. Compounds derived from this series bind to both human and rat GnRH receptors and antagonize GnRH-mediated increases in inositol phosphate production in cells containing recombinant human receptors. These compounds or their analogues may be useful as therapeutic agents for the treatment of hormone-dependent pathologies including prostate, breast and ovarian cancers.

Functional study of a recombinant form of human LHbeta-subunit variant carrying the Gly(102)Ser mutation found in Asian populations.

Genetic variants of human LH caused by amino acid replacements in the beta-subunit have been demonstrated to affect reproductive function. Occurrence of a G(1502)A substitution in the LHbeta gene leading to Gly(102)Ser replacement of the LHbeta protein has been found to be associated with infertility in the Singapore Chinese population. In the present study, a search for this LHbeta allele from 383 DNA samples from different continents, using a PCR-based strategy, demonstrated its total absence in these populations. Functional properties of the variant (V) (Gly(102)Ser substitution) LHbeta subunit were assessed using a recombinant (r) form of V-LH produced in HEK293 cells, in comparison with wild-type (WT) LH or hCG. The synthesized V-LH was purified by a single step of immunoaffinity chromatography, and it had a molecular weight of 30 kDa as determined by SDS-PAGE. The affinities of the WT-hCG and rV-LH in mouse Leydig tumour (mLT-1) cell LH receptor binding were similar, with K(d) values of 0.140 +/- 0.03 and 0.156 +/- 0.01 nmol/l respectively. Likewise, the effects of WT- and V-rLH preparations on mLT-1 cell cAMP and progesterone production were concentration-dependent and with similar biopotencies. In addition, HEK293 cells expressing the human LH receptor documented similar dose-dependent increases in inositol phosphate production by the two rLH forms. In conclusion, these findings demonstrate that Gly(102)Ser mutation of the LHbeta gene does not affect receptor binding and bioactivity of the hormone, when tested in vitro.

Protein–protein interactions regulate PTH1 receptor signalling

Association of Na+–H+ exchanger regulatory factors (NHERFs) with the parathyroid hormone 1 (PTH1) receptor via a PDZ-domain interaction leads to formation of molecular complexes that enhance Gi/o-mediated phospholipase C (PLC) activation but reduce Gs-mediated signalling.Evidence is accumulating that hormone receptors are present in the cells as part of multimolecular complexes that determine the function of the receptor. Mahon et al. [1xNa+/H+ exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling. Mahon, M.J. et al. Nature. 2002; 417: 858–861Crossref | PubMed | Scopus (228)See all References][1] have studied interactions of the C-terminal tail of the PTH1 receptor with other proteins, because earlier data indicated that the C-terminal tail modulates PTH1 receptor signalling. The authors performed yeast two-hybrid screens using the C-terminal tail of the PTH1 receptor as bait. In a human kidney cDNA library, they found that a fragment of Na+–H+ exchanger regulatory factor 2 (NHERF2) interacts strongly with the C-terminal tail of the PTH1 receptor. Because NHERF has been shown to interact with other hormone receptors, and it has two PDZ (PSD95, discs large protein, ZO1) domains that interact with well-defined C-terminal motifs, in addition to an ERM (ezrin, radixin, moesin)-binding motif, the authors have analysed the biological importance of this interaction. In vitro analyses revealed that the C-terminal end of the PTH1 receptor binds to the second PDZ domain (PDZ2) of NHERF2, and a similar interaction is detected with NHERF1. In PS120 Chinese hamster fibroblast cells expressing PTH1 receptor and NHERF2, coprecipitation of these molecules was observed. Most importantly, PTH-induced cAMP stimulation is inhibited when NHERF2 is coexpressed with the PTH1 receptor (from 10–15-fold stimulation without NHERF2 to only 2-fold stimulation with NHERF2), whereas the inositol phosphate (IP) response was markedly increased with NHERF2 coexpression (from 2–4-fold to 20–25-fold). The authors concluded that stimulation of IP production was mediated by Gi/o proteins, because the response was pertussis-toxin sensitive. Because all four isoenzymes of PLCβ, which mediate the hormone-induced generation of IP, contain consensus C-terminal PDZ domain interaction motifs, the authors have used pull-down assays to show that NHERF2 can form multiprotein complexes consisting of PLCβ1 bound to PDZ1 and PTH1 receptor bound to PDZ2. However, it remains to be elucidated whether other subtypes of PLCβ (e.g. PLCβ2) produce similar interactions. Furthermore, the authors have compared the PTH1 receptor-mediated signal generation in two cell lines. They show that in ECV304 cells (a human endothelium-derived cell line with abundant NHERF1 and 2 expression), PTH predominantly increases IP production, whereas in ROS 17/2.8 cells (a rat osteoblastic cell line with no detectable NHERF expression), PTH causes more than a 30-fold increase in cAMP production without a detectable effect on IP signal generation. The appearance of PLC-mediated signalling after transient transfection of NHERF2 into ROS17/2.8 cells was indirectly detected using an AP1-responsive luciferase reporter gene.These data demonstrate that expression of NHERFs leads to formation of molecular complexes that regulate the signalling of the PTH1 receptor. This might be particularly important in polarized cells, because the basal and luminal membranes often have different NHERF expression, and suggests that assembly of receptor complexes with NHERF and possibly other PDZ-domain-containing proteins could be a general mechanism for site-specific regulation of secondary messenger generation.

Identification of Domains Directing Specificity of Coupling to G-proteins for the Melanocortin MC3 and MC4 Receptors

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Conditionally expressed G alpha 15 couples to endogenous receptors in GH3 cells.

The mammalian G proteins G15 and G16 couple a wide variety of receptors to phospholipase C (PLC) in co-transfected systems, and it has been suggested that they can be used as tools in agonist-screening systems. Using the reversed tetracycline-controlled transactivation system we generated rat pituitary GH3 cell clones that expressed Galphal5 and Galpha16 conditionally to study the coupling of endogenous receptors to both G proteins. In cells expressing moderate levels of Galpha15, activation of various endogenous receptors increased inositol phosphate production, whereas conditional expression of Galpha16 had no significant effect on agonist-dependent PLC activity. Activation of PLC through Galpha15 in response to carbachol did not increase cytosolic [Ca2+] ([Ca2+]i) but stimulated protein kinase C. While carbachol decreased the secretory activity in non-induced GH3 cells, it increased secretion in cells expressing Galpha15. Our data demonstrate that Galpha15 has a higher functional promiscuity than Galpha16 when studied in a system that preserves physiological G protein and receptor levels. In addition, Galpha15-mediated coupling of a receptor to PLC can change the cellular response to receptor agonists, indicating that downstream cellular functions can be used to detect receptor activation in screening systems employing a promiscuous G protein.

Melanotrope cell plasticity: a key mechanism for the physiological adaptation to background color changes.

The intermediate lobe of the pituitary secretes the melanotropic hormone alpha-MSH, which in amphibians plays a crucial role in skin color adaptation. It has been previously demonstrated that, in the frog Rana ridibunda, the intermediate lobe is composed of two distinct subpopulations of melanotrope cells that can be separated in vitro by using Percoll density gradients. These two melanotrope cell subsets, referred to as high-density (HD) and low-density (LD) cells, differ in their ultrastructural characteristics as well as in their biosynthetic and secretory activity. However, the specific, physiological role of the heterogeneity displayed by melanotrope cells remains elusive. In the present study, we investigated the effects of background color adaptation on melanotrope cell subpopulations. We found that adaptation of frogs to dark or white environment did not modify either the overall number of cells per intermediate lobe or the apoptotic and proliferation rates of melanotrope cells. On the other hand, adaptation of the animals to a white background significantly increased the proportion of hormone-storage HD cells and caused a concomitant decrease in that of LD cells (which exhibit higher levels of alpha-MSH release and POMC messenger RNA than HD cells). Conversely, after black-background adaptation the proportion of LD cells was markedly increased, suggesting that interconversion of HD cells to LD cells occurs during physiological activation of the intermediate lobe. In addition, black-background adaptation also enhanced alpha-MSH release by both cell subpopulations and increased inositol phosphate production in LD cells. These data indicate that, in frog, the proportions of the two melanotrope cell subsets undergo marked modifications during skin color adaptation, likely reflecting the occurrence of a secretory cell cycle whose dynamics are highly correlated to the hormonal demand imposed by the environment.

Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness.

It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.

Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?

Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.