Yasser Saad

Activation of G-protein-coupled receptors: a common molecular mechanism

G-protein-coupled receptors (GPCRs) are a large family of proteins that contain a seven transmembrane helical structural motif. They mediate responses to several ligands by binding and activating intracellular heterotrimeric G proteins. Since the cloning of the first GPCR, insights gained from structure-function studies, genetics and drug development have contributed to uncovering a common mechanism that explains the activation of diverse GPCRs by their cognate agonists. This mechanism takes into consideration the conservation of the structure-function relationship in the basic seven transmembrane structural motif, and the dynamic changes in receptor conformation that are associated with activation. Combining models derived from the X-ray structure of rhodopsin with structure-function data allows a deeper understanding of the activation mechanism of GPCRs.

Interaction of Phe8 of Angiotensin II with Lys199 and His256 of AT1 Receptor in Agonist Activation

The acidic pharmacophores of selective ligands bind to Lys199 and His256 of the AT1 receptor (Noda, K., Saad, Y., Kinoshita, A., Boyle, T. P., Graham, R. M., Husain, A., and Karnik, S.(1995) J. Biol. Chem. 270, 2284-2289). In this report we examine how interactions between these residues and agonists activate inositol phosphate production in transiently transfected COS-1 cells. [Sar1] angiotensin (Ang II) II and [Sar1]Ang II-amide stimulated a 5-fold inositol phosphate response from wild-type AT1 receptor. The peptide antagonist [Sar1,Ile8]Ang II and the non-peptide agonist L-162,313 produced a partial but saturating response. Stimulation of wild-type receptor by [Sar1]Ang II-amide and the mutant K199Q and K199A receptors by [Sar1]Ang II demonstrates that AT1 receptor activation is not critically dependent on the ion-pairing of the α-COOH group of Ang II with Lys199. The mutation of His256 produced diminished inositol phosphate response without commensurate change in binding affinity of ligands. The His256 side chain is critical for maximal activation of the AT1 receptor, although isosteric Gln substitution is sufficient for preserving the affinity for Phe8-substituted analogues of [Sar1]Ang II. Therefore, AT1 receptor activation requires interaction of Phe8 side chain of Ang II with His256, which is achieved by docking the α-COOH group of Phe8 to Lys199. Furthermore, non-peptide agonists interact with Lys199 and His256 in a similar fashion.

Transducin-α C-terminal Peptide Binding Site Consists of C-D and E-F Loops of Rhodopsin

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and αt-(340-350), a peptide corresponding to the C terminus of the α subunit (αt) of bovine retinal G-protein, transducin. Direct binding of αt-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the αt C terminus of the transducin during light transduction in vivo In most G-proteins, the C termini of α subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.

Transcription Factors STAT6 and KLF4 Implement Macrophage Polarization via the Dual Catalytic Powers of MCPIP

Macrophage polarization plays a critical role in tissue homeostasis, disease pathogenesis, and inflammation and its resolution. IL-4–induced macrophage polarization involves induction of STAT6 and Krüppel-like factor 4 (KLF4), which induce each other and promote M2 polarization. However, how these transcription factors implement M2 polarization is not understood. We report that in murine macrophages MCP-1–induced protein (MCPIP), induced by KLF4, inhibits M1 polarization by inhibiting NF-κB activation and implements M2 polarization using both its deubiquitinase and RNase activities that cause sequential induction of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy required for M2 polarization. MCPIP also induces C/EBPβ and PPARγ, which promote M2 polarization. Macrophages from mice with myeloid-targeted overexpression of MCPIP show elevated expression of M2 markers and reduced response to LPS, whereas macrophages from mice with myeloid-specific deletion of MCPIP manifest elevated M1 polarization with enhanced phagocytic activity. Thus, both in vivo and in vitro experiments demonstrate that the transcription factors STAT6 and KLF4 implement IL-4–induced M2 polarization via the dual catalytic activities of MCPIP.

Participation of MCP-induced protein 1 in lipopolysaccharide preconditioning-induced ischemic stroke tolerance by regulating the expression of proinflammatory cytokines

BackgroundLipopolysaccharide (LPS) preconditioning-induced neuroprotection is known to be related to suppression of the inflammatory response in the ischemic area. This study seeks to determine if monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified CCCH Zn finger-containing protein, plays a role in focal brain ischemia and to elucidate the mechanisms of LPS-induced ischemic brain tolerance.MethodsTranscription and expression of MCPIP1 gene was monitored by qRT-PCR and Western blot. Mouse microglia was prepared from cortices of C57BL/6 mouse brain and primary human microglia was acquired from Clonexpress, Inc. Wild type and MCPIP1 knockout mice were treated with LPS (0.2 mg/kg) 24 hours before brain ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining.ResultsMCPIP1 protein and mRNA levels significantly increased in both mouse and human microglia and mouse brain undergoing LPS preconditioning. MCPIP1 mRNA level significantly increased in mice ipsilateral brain than that of contralateral side after MCAO. The mortality of MCPIP1 knockout mice was significantly higher than that of wild-type after MCAO. MCPIP1 deficiency caused significant increase in the infarct volume compared with wild type mice undergoing LPS preconditioning. MCPIP1 deficiency caused significant upregulation of proinflammatory cytokines in mouse brain. Furthermore, MCPIP1 deficiency increased c-Jun N terminal kinase (JNK) activation substantially. Inhibition of JNK signaling decreased the production of proinflammatory cytokines in MCPIP1 knock out mice after MCAO.ConclusionsOur data indicate that absence of MCPIP1 exacerbates ischemic brain damage by upregulation of proinflammatory cytokines and that MCPIP1 participates in LPS-induced ischemic stroke tolerance.

Congenic Interval Mapping of RNO10 Reveals a Complex Cluster of Closely-Linked Genetic Determinants of Blood Pressure

Genetic dissection of the rat genome for identifying alleles that cause abnormalities in blood pressure (BP) resulted in the mapping of a significant number of BP quantitative trait loci (QTLs). In this study we mapped at least one such BP QTL on rat chromosome 10 (RNO10) as being within the introgressed segment of a S.LEW congenic strain S.LEWx12x2x3x8 spanning 1.34 Mb from 70 725 437 bp to 72 063 232 bp. BP of 3 congenic strains that span shorter segments of this region was additionally examined. Results obtained indicate that LEW alleles that comprise a 375-kb introgressed segment of the congenic strain S.LEWx12x2x3x5 (70 725 437 bp to 71 100 513 bp) increase BP. The magnitude of change in BP exhibited by the 2 strains, S.LEWx12x2x3x8 and S.LEWx12x2x3x5, is the net phenotypic effect of the underlying genetic determinants of BP. In this respect, the current data are superior to previous QTL localization of BP QTL1, which was hypothesized based on differential congenic segments. Genetic dissection using these 2 congenic strains as tools is expected to facilitate further dissection of the regions. Meanwhile, differential congenic segments were used to predict and thereby prioritize regions for candidate gene analysis. Using this approach, 2 distinct regions of 401 kb and 409 kb within S.LEWx12x2x3x8 and a 104 kb region within S.LEWx12x2x3x5 were prioritized for further consideration. Because all of these genetic elements are located within a 1.06-Mb region of RNO10, our study has revealed a remarkable insight into a genomic module comprising very closely-linked, opposing genetic determinants of BP.

Angiotensinergic stimulation of vascular endothelium in mice causes hypotension, bradycardia, and attenuated angiotensin response

It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease.

Ecological Genetics of Tetrahymena thermophila: Mating Types, i‐Antigens, Multiple Alleles and Epistasis

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole‐genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV‐ and VII‐) and B(I‐) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A‐category genotype appears to lack mating type II, while one A‐category and all B‐category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B‐category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i‐antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i‐antigen. Molecular weight differences suggest that these paralogous i‐antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i‐ag frequencies suggest that i‐ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.

Reversible inactivation of AT2 angiotensin II receptor from cysteine–disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT2) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys35–Cys290 and Cys117–Cys195 disulfide bonds are formed in the wild‐type AT2 receptor. Disruption of the highly conserved Cys117–Cys195 disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys35–Cys290 bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT2 receptor‐specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re‐folded to high‐affinity state by DTT treatment. These results suggest that the free –SH group of Cys35 or Cys290 competes with the disulfide bond formation between Cys117 and Cys195. This Cys–disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non‐native disulfide bond.

Immobilization Antigen Variation in Natural Isolates of Tetrahymena thermophila

In Tetrahymena thermophila alternative forms of the major cell surface glycoprotein (the immobilization antigen) are specified by both allelic and non-allelic genes. The differential expression of non-allelic genes is affected primarily by temperature and culture medium. This report describes expression and genetic variation of immobilization antigens among 2,600 clones isolated from natural populations. The temperature regulated L (<20 o ) and H (20 o C-36 o C) antigens and two new antigens J and K were present among ∼57% of isolates; the remaining 43% appear to have unknown antigens. Genetic and Southern analyses show that J and K are due to genes with dominant epistasis over H and that the gene for K is also epistatic over that for J. This is the first reported instance of naturally occurring epistasis involving immobilization antigen expression in T. thermophila. In ponds, the frequencies of J and H vary inversely in a manner consistent with dominant epistasis. The frequencies of J and H also show seasonal variation, with J more common in the late spring, and H more common in the late summer and fall. L and H (and J) also show seasonal variation, with L more common in the early spring and late fall. Allelic variation was also found among the H antigens. Immunodiffusion showed that the H3 protein of natural isolates is partially identical to H3 of inbred strain B. In addition, two Hind III restriction fragment length polymorphisms were found among the natural SerH3 genes. New SerH3 genes also appeared to segregate in crosses. The genetic and seasonal variation in i-antigen frequencies suggests an important biological role for these surface proteins