Comprehensive biochemical analysis of rare prostacyclin receptor variants: study of association of signaling with coronary artery obstruction

Currently, pharmacogenetic studies are at an impasse as the low prevalence (<2%) of most variants hinder their pharmacogenetic analysis with population sizes often inadequate for sufficiently powered studies. Grouping rare mutations by functional phenotype rather than mutation site can potentially increase sample size. Using human population-based studies (n = 1,761) to search for dysfunctional human prostacyclin receptor (hIP) variants, we recently discovered 18 non-synonymous mutations, all with frequencies less than 2% in our study cohort. Eight of the 18 had defects in binding, activation, and/or protein stability/folding. Mutations (M113T, L104R, and R279C) in three highly conserved positions demonstrated severe misfolding manifested by impaired binding and activation of cell surface receptors. To assess for association with coronary artery disease, we performed a case-control study comparing coronary angiographic results from patients with reduced cAMP production arising from the non-synonymous mutations (n = 23) with patients with non-synonymous mutations that had no reduction in cAMP (n = 17). Major coronary artery obstruction was significantly increased in the dysfunctional mutation group in comparison with the silent mutations. We then compared the 23 dysfunctional receptor patients with 69 age- and risk factor-matched controls (1:3). This verified the significantly increased coronary disease in the non-synonymous dysfunctional variant cohort. This study demonstrates the potential utility of in vitro functional characterization in predicting clinical phenotypes and represents the most comprehensive characterization of human prostacyclin receptor genetic variants to date.     

Impaired receptor binding and activation associated with a human prostacyclin receptor polymorphism

The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structure-function characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4, K(i) = 2.2 +/- 1.2 nm; pH 6.8 K(i) = 45.6 +/- 12.0 nm). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC(50) = 2.8 +/- 0.5 nm versus wild-type hIP EC(50) = 0.5 +/- 0.1 nm; pH 6.8, R212H EC(50) = 3.2 +/- 1.6 nm versus wild-type hIP EC(50) = 0.5 +/- 0.2 nm). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP.     

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood. Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl-terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaprost stimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11^S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val²⁹⁹–Gln³²⁰) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.     

Identification of a novel endoplasmic reticulum export motif within the eighth α-helical domain (α-H8) of the human prostacyclin receptor

The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val(299)-Val(307) within the eighth helical domain (α-H8) adjacent to the palmitoylated residues at Cys(308)-Cys(311). However, the factors determining the anterograde transport of the newly synthesised hIP from the endoplasmic reticulum (ER) to the plasma membrane (PM) have not been identified. The aim of the current study was to identify the major ER export motif(s) within the hIP initially by investigating the role of Lys residues in its maturation and processing. Through site-directed and Ala-scanning mutational studies in combination with analyses of protein expression and maturation, functional analyses of ligand binding, agonist-induced intracellular signalling and confocal image analyses, it was determined that Lys(297), Arg(302) and Lys(304) located within α-H8 represent the critical determinants of a novel ER export motif of the hIP. Furthermore, while substitution of those critical residues significantly impaired maturation and processing of the hIP, replacement of the positively charged Lys with Arg residues, and vice versa, was functionally permissible. Hence, this study has identified a novel 8 residue ER export motif within the functionally important α-H8 of the hIP. This ER export motif, defined by "K/R(X)(4)K/R(X)K/R," has a strict requirement for positively charged, basic Lys/Arg residues at the 1st, 6th and 8th positions and appears to be evolutionarily conserved within IP sequences from mouse to man.     

Association of P2Y2 receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

The P2Y₂ receptor is a G-protein-coupled receptor with adenosine 5′-triphosphate (and UTP) as natural ligands. It is thought to be involved in bone physiology in an anti-osteogenic manner. As several non-synonymous single nucleotide polymorphisms (SNPs) have been identified within the P2Y₂ receptor gene in humans, we examined associations between genetic variations in the P2Y₂ receptor gene and bone mineral density (BMD) (i.e., osteoporosis risk), in a cohort of fracture patients. Six hundred and ninety women and 231 men aged ≥50 years, visiting an osteoporosis outpatient clinic at Maastricht University Medical Centre for standard medical follow-up after a recent fracture, were genotyped for three non-synonymous P2Y₂ receptor gene SNPs. BMD was measured at three locations (total hip, lumbar spine, and femoral neck) using dual-energy X-ray absorptiometry. Differences in BMD between different genotypes were tested using analysis of covariance. In women, BMD values at all sites were significantly different between the genotypes for the Leu46Pro polymorphism, with women homozygous for the variant allele showing the highest BMD values (0.05 > p > 0.01). The Arg312Ser and Arg334Cys polymorphisms showed no differences in BMD values between the different genotypes. This is the first report that describes the association between the Leu46Pro polymorphism of the human P2Y₂ receptor and the risk of osteoporosis.     

Structure-function analysis of helix 8 of human calcitonin receptor-like receptor within the adrenomedullin 1 receptor

Adrenomedullin 1 (AM₁) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ∼20% reduction in specific ¹²⁵I-AM binding but little change in their IC₅₀ values. Both mutants showed 6-8-fold higher EC₅₀ values for AM-induced cAMP production and ∼50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ∼45%, but surface expression and ¹²⁵I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM₁ receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM₁ receptor internalization, which is not dependent on Gs coupling.     

Clusters of transmembrane residues are critical for human prostacyclin receptor activation

Relaxation of vascular smooth muscle and prevention of blood coagulation are mediated by ligand-induced activation of the human prostacyclin (hIP) receptor, a seven-transmembrane-domain G-protein-coupled receptor (GPCR). In this study, we elucidate the molecular requirements for receptor activation within the region of the ligand-binding pocket, identifying transmembrane residues affecting potency. Eleven of 30 mutated residues in the region of the ligand-binding domain exhibited defective activation (decreased potency). These critical residues localized to four distinct clusters (analysis via a rhodopsin-based human prostacyclin receptor homology model). Residues Y75(2.65) (TMII), F95(3.28) (TMIII), and R279(7.40) (TMVII) comprised the immediate binding-pocket cluster and were shown to be essential for proper receptor activation, compared to equivalent expression levels of the wild-type hIP (WT EC(50) = 1.2 +/- 0.1 nM; Y75(2.65)A EC(50) = 347.3 +/- 62.8 nM, p < 0.001; F95(3.28)A EC(50) = 8.0 +/- 0.6 nM, p < 0.001; R279(7.40)A EC(50) = 130 +/- 63.0 nM, p < 0.001). Residues S20(1.39) (TMI), F24(1.43) (TMI), and F72(2.62) (TMII) were localized to a cluster involving P17(1.36), a critical residue thought to facilitate transmembrane movement during changes in activation conformation. A third cluster formed around amino acid D60(2.50) (TMII), containing the highly conserved (100% of prostanoid receptors) D288(7.49)/P289(7.50) motif located in TMVII. Last, a large hydrophobic cluster composed of aromatic residues F146(4.52) (TMIV), F150(4.56) (TMIV), F184(5.40) (TMV), and Y188(5.44) (TMV) was observed away from the ligand-binding pocket, but still necessary for hIP activation. These results assist in delineating the potential molecular requirements for agonist-induced signaling through the transmembrane domain. Such observations may be generally applicable, as many of these clusters are highly conserved among the prostanoid receptors as well as other class A GPCRs.     

Genetic diversity of the toll-like receptor 2 (TLR2) in hare (Lepus capensis) populations from Tunisia

Toll-like receptors (TLRs) are a major group of proteins that recognize molecular components of infectious agents, known as pathogen associated molecular patterns (PAMPs). The structure of these genes is similar and characterized by the presence of an ectodomain, a signal transmembrane segment and a highly conserved cytoplasmic domain. The latter domain is homologous to the human interleukin-1 receptor (IL1R) and human IL-18 receptor (IL-18R) and designated TIR domain. The latter domain of the TLR genes was suggested to be very conservative and its evolution is driven by purifying selection. Variability and evolution of the TIR sequences of TLR2 gene were studied in three hare populations from Tunisia with different ecological characteristics (NT–North Tunisia with Mediterranean, CT–Central Tunisia with semi-arid, and ST–South Tunisia with arid climate). Sequencing of a 372 bp fragment of TIR2 revealed 25 alleles among 110 hares. Twenty variable nucleotide positions were detected, of which 7 were non-synonymous. The highest variability was observed in CT, with 16 polymorphic positions. In ST, only 4 polymorphic nucleotide positions were detected with all diversity values lower than those recorded for the other two populations. By using several approaches, no positive selection was detected. However, evidence of purifying selection was found at two positions. The logistic models of the most common TIR2 protein variant that we run to examine whether its occurrence was affected by climatic variation independent of the geographic sample location suggested only a longitudinal effect. Finally, the mapping of the non-synonymous mutations to the inferred tertiary protein structure showed that they were all localized in the different loop regions. Among all non-synonymous substitutions, three were suggested to be deleterious as evidenced by PROVEAN analysis. The observed patterns of variability characterized by low genetic diversity in ST might suggest that the TIR region was more affected, than other markers, by genetic drift or/and that these patterns were shaped by different selective pressures under different ecological conditions. Notably, this low diversity was not detected by other (putatively neutral) microsatellite markers analysed in the course of other studies. But low diversity was also found for two MHC class II adaptive immune genes. As expected from functionally important regions, the evolution of the TIR2 domain is mainly driven by purifying selection. However, the occurrence of deleterious non-synonymous substitutions might highlight the flexible evolution of the TIR genes and/or their interactions with other proteins.     

Molecular docking studies of 1-(substituted phenyl)-3-(naphtha [1, 2-d] thiazol-2-yl) urea/thiourea derivatives with human adenosine A(2A) receptor

Computational assessment of the binding interactions of drugs is an important component of computer-aided drug design paradigms. In this perspective, a set of 30 1-(substituted phenyl)-3-(naphtha[1, 2-d] thiazol-2-yl) urea/thiourea derivatives showing antiparkinsonian activity were docked into inhibitor binding cavity of human adenosine A(2A) receptor (AA2AR) to understand their mode of binding interactions in silico. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.483) between docking score and antiparkinsonian activity (in terms of % reduction in catalepsy score). Potent antiparkinsonian agents carried methoxy group in the phenyl ring, exhibited both hydrophilic and lipophilic interactions with lower energy of binding at the AA(2A)R. These molecular docking analyses should, in our view, contribute for further development of selective AA(2A)R antagonists for the treatment of Parkinson's disease.     

Variation in the human TAS1R taste receptor genes

We have performed a comprehensive evaluation of single-nucleotide polymorphisms (SNPs) and haplotypes in the human TAS1R gene family, which encodes receptors for sweet and umami tastes. Complete DNA sequences of TAS1R1-, TAS1R2-, and TAS1R3-coding regions, obtained from 88 individuals of African, Asian, European, and Native American origin, revealed substantial coding and noncoding diversity: polymorphisms are common in these genes, and polymorphic sites and SNP frequencies vary widely in human populations. The genes TAS1R1 and TAS1R3, which encode proteins that act as a dimer to form the umami (glutamate) taste receptor, showed less variation than the TAS1R2 gene, which acts as a dimer with TAS1R3 to form the sweet taste receptor. The TAS1R3 gene, which encodes a subunit common to both the sweet and umami receptors, was the most conserved. Evolutionary genetic analysis indicates that these variants have come to their current frequencies under natural selection during population growth and support the view that the coding sequence variants affect receptor function. We propose that human populations likely vary little with respect to umami perception, which is controlled by one major form of the receptor that is optimized for detecting glutamate but may vary much more with respect to sweet perception.     

Agonists can discriminate between cloned human and mouse prostacyclin receptors

The ability of prostacyclin analogues to stimulate adenylyl cyclase (AC) and phospholipase C (PLC) in Chinese hamster ovary (CHO) cells expressing cloned human (hIP) or cloned mouse (mIP) prostacyclin receptors has been compared. For hIP, the order of potency (pEC(50)) for stimulating AC and PLC pathways was similar: AFP-07 (9.3, 8.4)>cicaprost (8.3, 6.9), iloprost (7.9, 6.8)>taprostene (7.4, 6.8)>carbacyclin (6.9, 6.6), PGE(1) (6.6, 5.1). Although the standard IP agonists cicaprost and iloprost behaved similarly in both hIP and mIP receptor-expressing cells, carbacyclin and PGE(1) showed significantly higher potency at the mIP receptor, suggesting that the agonist recognition sites on hIP and mIP receptors are not identical. A further distinction between hIP and mIP receptors was found with taprostene, which had greater efficacy at hIP receptors (AC 94%, PLC 14%) than at mIP receptors (AC 77%, PLC 0%) (cicaprost=100% in each assay).     

Agonist-dependent internalization and trafficking of the human prostacyclin receptor: a direct role for Rab5a GTPase

The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5a(S34N) did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5a(Q79L) significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.     

Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Galpha(s) and Galpha(16) hybrid signaling

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G(s) and G(q) proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr(705) and Ser(727) phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cbeta, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Galpha(16)-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr(705) phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser(727) phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G(16)-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G(s) pathway. This postulation was confirmed by expressing constitutively active Galpha(16)QL and Galpha(s)QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Galpha(16)QL-induced STAT3 phosphorylations could be converted by the mere presence of Galpha(s)QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Galpha(16)-mediated inhibitor-sensitivity upon cicaprost induction in Galpha(s)-knocked down HEL cells again verified the pivotal role of G(s) signal. Taken together, our observations illustrate that co-stimulation of G(s) and G(q) can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.     

Regulation of the human prostacyclin receptor gene by the cholesterol-responsive SREBP1

Prostacyclin and its prostacyclin receptor, the I Prostanoid (IP), play essential roles in regulating hemostasis and vascular tone and have been implicated in a range cardio-protective effects but through largely unknown mechanisms. In this study, the influence of cholesterol on human IP [(h)IP] gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol depletion increased human prostacyclin receptor (hIP) mRNA, hIP promoter-directed reporter gene expression, and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active sterol-response element binding protein (SREBP)1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression, and deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, and immunofluorescence microscopy corroborated that cholesterol depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol depletion, which occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression, which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of IP expression within the human vasculature.     

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G_α subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A_2B receptor (hA_2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA_2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: G_αi, G_αs, G_αq, and G_α12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G_α subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103^3.50 and I107^3.54 are vital too in G protein-coupling and the activation of the hA_2BR, whereas L213^IL3 is more important in G protein inactivation. Substitution of S235^6.36 to alanine provided the most divergent G protein-coupling profile. Finally, L236^6.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.     

Association of the melanocortin 4 receptor with feed intake and daily gain in F2 Mangalitsa x Piétrain pigs

The melanocortin 4 receptor (MC4R) is a key factor in the regulation of energy balance and body weight. Hence it is a candidate for feed intake and energy homeostasis-related traits. Studies in humans and swine have revealed several sequence variants in the gene that are associated with some of these traits. In pigs the coding non-synonymous missense variant Asp298Asn in MC4R has been associated with feed intake, fatness and growth. Here we confirm the association of this Piétrain-derived polymorphism with feed intake and daily gain in the F2 generation of a Mangalitsa x Piétrain cross. In one Piétrain founder animal, we detected an additional non-synonymous missense variant Arg236His. Thus, the MC4R gene could be a useful marker for increased growth in the relatively slow-growing Piétrain breed.     

Detecting ligand interactions with G protein-coupled receptors in real-time on living cells

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand–GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.     

Constitutive dimerization of human serotonin 5-HT4 receptors in living cells

Serotonin 5-HT4 receptor isoforms are G protein-coupled receptors (GPCRs) with distinct pharmacological properties and may represent a valuable target for the treatment of many human disorders. Here, we have explored the process of dimerization of human 5-HT4 receptor (h5-HT4R) by means of co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Constitutive h5-HT4(d)R dimer was observed in living cells and membrane preparation of CHO and HEK293 cells. 5-HT4R ligands did not influence the constitutive energy transfer of the h5-HT4(d)R splice variant in intact cells and isolated plasma membranes. In addition, we found that h5-HT4(d)R and h5-HT4(g)R which structurally differ in the length of their C-terminal tails were able to form constitutive heterodimers independently of their activation state. Finally, we found that coexpression of h5-HT4R and beta2-adrenergic receptor (beta2AR) led to their heterodimerization. Given the large number of h5-HT4R isoforms which are coexpressed in a same tissue, our results points out the complexity by which this 5-HTR sub-type mediates its biological effects.     

Investigation of a functional requirement for isoprenylation by the human prostacyclin receptor.

In the current study, we have established that the human (h) prostacyclin receptor (IP) is isoprenylated in whole cells. Through site directed mutagenesis and generation of the isoprenylation defective hIPSSLC, it was established that while isoprenylation of hIP does not influence ligand binding, it is obligatory for agonist activation of adenylyl cyclase and cAMP generation. Overexpression of GalphaS significantly augmented cAMP generation by the hIP but not by the hIPSSLC. Moreover, GalphaS co-immunoprecipitated with hIP following agonist activation but did not co-immunoprecipitate with hIPSSLC. Whereas hIP mediated concentration-dependent activation of phospholipase C (PLC); the extent of PLC activation by hIPSSLC was impaired compared to hIP. Co-expression of Galphaq significantly augmentated intracellular calcium mobilization by the hIP but not by hIPSSLC. Moreover, whereas Galphaq co-immunoprecipitated with hIP, it failed to co-immunoprecipitate with hIPSSLC. While both the hIP and hIPSSLC underwent agonist-induced internalization, the kinetics and extent of hIPSSLC internalization was impaired compared to hIP. Altering the CAAX motif of the hIP from a farnesyl (-CSLC) to a geranylgeranyl (-CSLL) isoprene acceptor, to generate hIPCSLL, did not affect ligand binding and yielded a receptor that exhibited identical signalling through both Gs- and Gq-coupled effectors to that of hIP. Thus, whereas isoprenylation of hIP does not influence ligand binding, it is functionally imperative in regulating post-receptor events including agonist-activation of adenylyl cyclase, for efficient activation of PLC and for receptor internalization. Though the nature of the isoprenoid attached to hIP does not act as a major determinant, the presence of an isoprenoid group, for example farnesyl or geranylgeranyl, is required for functional receptor-G protein interaction and coupling and for efficient agonist- induced receptor internalization.