Gaétan Guillemette

Regulation of VEGF‐induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways

Vascular endothelial growth factor (VEGF) is a potent angiogenic and inflammatory mediator. We have recently shown that this latter effect requires the activation of Flk‐1 receptor and subsequent endothelial cell (EC) PAF synthesis. However, the intracellular events that regulate EC PAF synthesis upon Flk‐1 stimulation by VEGF remain to be elucidated. Using specific inhibitors and Western blot analysis, we herein report that in bovine aortic endothelial cells (BAEC), VEGF induces the synthesis of PAF through the cascade activation of Flk‐1 receptor, phospholipase Cγ (PLCγ), protein kinase C (PKC) and p42/44 mitogen‐activated protein kinases (MAPK). Moreover, we demonstrate that VEGF‐mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso‐PAF to PAF. Interestingly, we observed that VEGF also promoted the activation of the phosphatidyl inositol‐3‐phosphate kinase (PI3K) pathway, and that its blockade potentiated PAF synthesis following a VEGF treatment. Consequently, it appears that the PI3K pathway acts as a negative regulator of EC PAF synthesis. Taken together, these results allow a better understanding of the intracellular events activated upon EC stimulation by VEGF, and shed a new light on the mechanisms by which VEGF induces PAF synthesis.

Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT(1) receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine-scanning mutagenesis

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket. Each residue within the Ile103–Tyr127 region was mutated one at a time to a cysteine. Treating the A104C, N111C, and L112C mutant receptors with the charged sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA) strongly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD3 reporter cysteines engineered in a constitutively active AT1 receptor. Indeed, two additional mutants (S109C and V116C) were found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT1 receptor causes a minor counterclockwise rotation of TMD3, thereby exposing residues, which are not present in the inactive state, to the binding pocket. This pattern of accessibility of residues in the TMD3 of the AT1 receptor parallels that of homologous residues in rhodopsin. This study identified key elements of TMD3 that contribute to the activation of class A G protein-coupled receptors through structural rearrangements.

Photolabelling the rat urotensin II/GPR14 receptor identifies a ligand-binding site in the fourth transmembrane domain.

A urotensin II (U-II) peptide analogue containing the photoreactive p -benzoyl-L-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe(6)]U-II bound the receptor expressed in COS-7 cells with high affinity (IC(50) 0.7 nM) and was as potent as U-II in the agonist-induced production of inositol phosphate. Photolabelling of the U-II receptor with (125)I-[Bz-Phe(6)]U-II resulted in the specific formation of a glycosylated (125)I-[Bz-Phe(6)]U-II-U-II receptor complex of 60 kDa. Digestion of the 60 kDa complex with endoproteinase Glu-C generated a fragment of 17 kDa circumscribing the labelled fragment to residues 148-286. Digestion of the ligand-receptor complex with endoproteinase Arg-C produced a short peptide of 4 kDa corresponding to fragments 125-148, 167-192 or 210-233. CNBr treatment of the endoproteinase-Glu-C and -Arg-C fragments yielded 2 kDa fragments, defining the labelling site to methionine residues 184/185 of the fourth transmembrane domain. Photolabelling of two mutant receptors, M184L/M185L and M184A/M185A, led to a significant decrease in the overall yield of covalent labelling. Taken together, our results indicate that position 6 of U-II normally occupied by phenylalanine would interact with Met(184) and/or Met(185) of the fourth transmembrane domain of the U-II receptor. This information should be of significant value in the study of the interactions between U-II and its cognate receptor.

Identification of Angiotensin II-binding Domains in the Rat AT2 Receptor with Photolabile Angiotensin Analogs

To identify binding domains between angiotensin II (AngII) and its type 2 receptor (AT2), two different radiolabeled photoreactive analogs were prepared by replacing either the first or the last amino acid in the peptide with p-benzoyl-L-phenylalanine (Bpa). Digestion of photolabeled receptors with kallikrein revealed that the two photoreactive analogs label the amino-terminal part of the receptor within the first 182 amino acids. Digestion of 125I-[Bpa1]AngII·AT2 receptor complex with endoproteinase Lys-C produced a glycoprotein of 80 kDa. Deglycosylation of this 80-kDa product decreased its apparent molecular mass to 4.6 kDa and further cleavage of this 4.6-kDa product with V8 protease decreased its molecular mass to 3.6 kDa, circumscribing the labeling site of 125I-[Bpa1]AngII within amino acids 3-30 of AT2 receptor. Treatment of 125I-[Bpa8]AngII·AT2 receptor complex with cyanogen bromide produced two major receptor fragments of 3.6 and 2.6 kDa. Cyanogen bromide hydrolysis of a mutant AT2 receptor produced two major fragments of 12.6 kDa and 2.6 kDa defining the labeling site of 125I-[Bpa8]AngII within residues 129-138 of AT2 receptor. Our results indicate that the amino-terminal tail of the AT2 receptor interacts with the amino-terminal end of AngII, whereas the inner half of the third transmembrane domain of AT2 receptor interacts with the carboxyl-terminal end of AngII.

Immunohistochemical localization of type 2 inositol 1,4,5‐trisphosphate receptor to the nucleus of different mammalian cells

The inositol 1,4,5‐trisphosphate receptor (InsP3R) is a ligand‐gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti‐InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS‐7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co‐localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity‐binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed. J. Cell. Biochem. 85: 219–228, 2002.

METHIONINE PROXIMITY ASSAY, A NOVEL METHOD FOR EXPLORING PEPTIDE LIGAND–RECEPTOR INTERACTION

ABSTRACT Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p, p″-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.

Biological properties and functional determinants of the urotensin II receptor

The urotensin II receptor (UT) is a member of the G protein-coupled receptor (GPCR) family and binds the cyclic undecapeptide urotensin II (U-II) as well as the octapeptide urotensin II-related peptide (URP). The active UT mediates pleiotropic effects through various signal transduction pathways, including coupling to G proteins and activating the mitogen-activated protein kinase pathway. Several highly conserved residues and motifs of class A GPCRs that are important for activity are found in UT. This review highlights some of the putative roles of these motifs in the binding, activation and desensitization of UT.

N- and C-terminal structure-activity study of angiotensin II on the angiotensin AT2 receptor

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV >> Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.

S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II

1 Nitric oxide (NO) is known to affect the properties of various proteins via the S‐nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK‐293 cells. 2 SNP dose‐dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. 3 The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPγS or the soluble guanylyl cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one. 4 The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. 5 These results suggest that NO decreased the binding affinity of the AT1 receptor by S‐nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II.