Ying-Hong Feng

A Truncated P2X7 Receptor Variant (P2X7-j) Endogenously Expressed in Cervical Cancer Cells Antagonizes the Full-length P2X7 Receptor through Hetero-oligomerization

A truncated naturally occurring variant of the human receptor P2X7 was identified in cancer cervical cells. The novel protein (P2X7-j), a polypeptide of 258 amino acids, lacks the entire intracellular carboxyl terminus, the second transmembrane domain, and the distal third of the extracellular loop of the full-length P2X7 receptor. The P2X7-j was expressed in the plasma membrane; it showed diminished ligand-binding and channel function capacities and failed to form pores and mediate apoptosis in response to treatment with the P2X7 receptor agonist benzoyl-ATP. The P2X7-j interacted with the full-length P2X7 in a manner suggesting heterooligomerization and blocked the P2X7-mediated actions. Interestingly, P2X7-j immunoreactivity and mRNA expression were similar in lysates of human cancer and normal cervical tissues, but fulllength P2X7 immunoreactivity and mRNA expression were higher in normal than in cancer tissues, and cancer tissues lacked 205-kDa P2X7 immunoreactivity suggesting lack of P2X7 homo(tri)-oligomerization. These results identify a novel P2X7 variant with apoptosis-inhibitory actions, and demonstrate a distinct regulatory property for a truncated variant to antagonize its full-length counterpart through hetero-oligomerization. This may represent a general paradigm for regulation of a protein function by its variant.

Role of Aromaticity of Agonist Switches of Angiotensin II in the Activation of the AT1 Receptor

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996)Biochemistry 35, 16435–16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.

The P2X7 Receptor: A Novel Biomarker of Uterine Epithelial Cancers

Objective: To determine expression of the P2X7 receptor in normal and in cancer uterine tissues. The rationale was that the receptor P2X7 regulates constitutive apoptosis in uterine epithelial cells, and previous studies showed diminished P2X7-mediated apoptosis in cancer uterine cells compared with normal cells. Methods: A clinical, experimental feasibility study. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women ages 25 to 75. End points for P2X7 mRNA were quantitative PCR and in situ hybridization, and end points for P2X7 protein were Western blots and immunostaining using anti-P2X7 antibody. Results: (a) In normal uteri, P2X7 mRNA and protein were expressed predominantly in the epithelial (endometrial, endocervical, and ectocervical) cells. (b) Expression of the P2X7 mRNA and protein was absent from endometrial and endocervical adenocarcinoma tissues and from cervical squamous cell carcinoma tissues. (c) In cervical dysplasia, P2X7 protein was absent in the dysplastic lesions. (d) Semiquantitative analysis using P2X7 mRNA (normalized in each tissue to the constitutive glyceraldehyde-3-phosphate dehydrogenase) and P2X7 protein levels (normalized in each tissue to the constitutive tubulin) revealed that P2X7 mRNA and/or protein levels can distinguish uterine normal from cancer tissues at high degrees of sensitivity (92%, 100%) and specificity (100%, 90%). Summary and Conclusions: (a) Levels of the P2X7 are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. (b) The data suggest that tissue P2X7 mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues. (Cancer Epidemiol Biomarkers Prev 2006;15(10):1906–13)

Unconventional Homologous Internalization of the Angiotensin II Type-1 Receptor Induced by G-Protein–Independent Signals

Internalization of a G-protein–coupled receptor (GPCR) is essential to the desensitization, endocytosis, and signal transduction of the receptor. It has been the general view that conventional homologous internalization of a GPCR requires activation of the G-protein(s) coupled to the receptor. However, whether and how GPCR-mediated G-protein–independent signals trigger receptor internalization remains unknown, although G-protein–independent internalization has been reported. Here we show that an angiotensin II (Ang II) type-1 (AT1) receptor mutant incapable of activating any G-protein still undergoes normal internalization. Substitution of Asp125 with Ala and Arg126 with Leu at the highly conserved DRY motif of the AT1 receptor disabled the ability of the receptor to activate G-proteins, as shown by various Ang II binding studies, GDP–GTP exchange, and inositol phosphate production assays. Surprisingly, the mutant internalized normally in the presence of Ang II and transactivated the epidermal growth factor receptor (EGFR). Similar to the wild-type receptor, overexpression of a dominant-negative K220R mutant GRK2 diminished the internalization of D125A-R126L but not the transactivation of EGFR. These data indicate that G-protein–independent specific signals may also trigger homologous internalizations of the AT1 receptor through β-arrestin–dependent and –independent pathways, suggesting a possible mechanism for G-protein–independent activation of G-protein–coupled receptor kinases (GRKs). This may represent a general mechanism for triggering GPCR internalization.

Nicotine Induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming.

Endogenously Expressed Truncated P2X7 Receptor Lacking the C-Terminus is Preferentially Upregulated in Epithelial Cancer Cells and Fails to Mediate Ligand-Induced Pore Formation and Apoptosis

A truncated naturally occurring variant of the human purinergic receptor P2X7 (P2X7-R) was found in human cancer cervical cells. The novel protein consists of 258 amino acids, and compared to the wild-type P2X7-R it lacks the entire intracellular carboxy terminus, the second transmembrane domain, and the distal third of the extracellular loop. The truncated P2X7-R failed to form pores and mediate apoptosis, and it interacted with the wild-type P2X7-R in a manner suggesting auto-hetero-oligomerization. In contrast to cancer cells the novel truncated P2X7-R was expressed relatively little in normal cervical cells. These data raise the possibility that coexpression of the truncated form could block P2X7 mediated apoptosis and promote uncontrolled growth of cells.

Role of transmembrane helix IV in G-protein specificity of the angiotensin II type 1 receptor.

G-protein activation by G-protein coupled receptors (GPCRs) is accomplished through proper interaction with the cytoplasmic loops rather than through sequence-specific interactions. However, the mechanism by which a specific G-protein is selected by a GPCR is not known. In the current model of GPCR activation, agonist binding modulates helix-helix interactions, which is necessary for fully determining G-protein specificity and stimulation of GDP/GTP exchange. In this study, we report that a single-residue deletion in transmembrane helix IV leads the angiotensin II type 1 (AT1) receptor chimera CR17 to retain GTP-sensitive high affinity for the agonist angiotensin II but results in complete inactivation of intracellular inositol phosphate production. The agonist dissociation profile of CR17 in the presence of guanosine 5′-3-O-(thio)triphosphate suggests that the activation-induced conformational changes of the chimeric receptor itself remain intact. Insertion of an alanine at position 149 (CR17▿149A) in this chimera rescued the inactive phenotype, restoring intracellular inositol phosphate production by the chimera. This finding suggests that in the wild-type AT1 receptor the orientation of transmembrane helix IV-residues following Cys149 is a key determinant for effectively distinguishing among various structurally similar G-proteins. The results emphasize that the contacts within the membrane-embedded portion of transmembrane helix IV in the AT1 receptor is important for specific G-protein selection.

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.

Expression and activation of the reprogramming transcription factors

Recently, a series of exciting reports have revealed that terminally differentiated somatic cells can be reprogrammed to generate induced pluripotent stem (iPS) cells via overexpression of a cocktail of transcription factors such as Oct3, Sox2, Klf4, and c-Myc or Oct3, Sox2, Nanog, and Lin28. Most recently, these iPS cells has been used to generate viable, live-born progeny by tetraploid complementation. Reprogramming of iPS cells inaugurates a new era of biology and medicine, it inevitably brings new challenges, e.g., how these factors induce reprogramming and how their expression is regulated. To facilitate iPS cell research, this review focuses on how expression and activation of these transcription factors are regulated.

High glucose instigates tubulointerstitial injury by stimulating hetero-dimerization of adiponectin and angiotensin II receptors

Abnormal expression and dysfunction of adiponectin and the cognate receptors are involved in diabetes and diabetic kidney disease (DKD), whereas angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) alleviate diabetic albuminuria and prevent development of DKD through upregulation of adiponectin expression. Here we report that high glucose stimulates expression of angiotensin II (AngII) receptors (AT1 and AT2) in renal proximal tubular epithelial cells (NRK-52E). These receptors underwent hetero-dimerization with adiponectin receptor AdipoR1 and AdipoR2, respectively. High glucose inhibited the dimerization between AT1 and AT2. Interestingly, these hetero-dimers instigated tubulointerstitial injury by inhibiting the cytoprotective action of the adiponectin receptors. These modes of receptor-receptor hetero-dimerization may contribute to high glucose-induced renal tubulointerstitial injury and could be potential therapeutic targets.