Somatostatin-induced SSTR2A endocytosis is regulated through β-arrestin2Arf6-EFA6A-PLD1 cascade in pancreatic β-cells (RINm5F) by Dai Tan Vo A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biomedical Sciences (Pharmacology) Program of Study Committee: Walter H Hsu, Major Professor Steven Carlson Timothy A Day Anumantha G Kanthasamy Michael J Kimber Richard J Martin Iowa State University Ames, Iowa 2011 ii   TABLE OF CONTENTS ABSTRACT iii LIST OF ABBREVIATIONS iv CHAPTER 1: GENERAL INTRODUCTION CHAPTER 2: MATERIALS AND METHODS 22 CHAPTER 3: RESULTS 30 CHAPTER 4: DISCUSSION 66 REFERENCES 74 ACKNOWLEDGEMENTS 93 iii     ABSTRACT Somatostatin (SS) receptors (SSTRs) undergo endocytosis following agonist stimulation We investigated the mechanism underlying SS-induced SSTR2A endocytosis The results from our previous work demonstrated that SS increases phospholipase D (PLD1) activity through Gβγ leading to the increase in phosphatidylinositol 4,5-bisphosphate (PIP2) levels in clonal β-cells HIT-T15 We also demonstrated that Arf6 and EFA6A are involved in the SS-induced PLD activity in HIT-T15 In the present study, we found that in clonal β-cells (RINm5F) overexpressing SSTR2A, SS-14 evoked endocytosis of these receptors By using radioligand binding techniques, we found that β-arrestin2, the ADP/ATP exchange factor for Arf6 A (EFA6A), ADP-ribosylation factor (Arf6), and PLD1 partcipated in the SS-induced SSTR2A endocytosis In addition, immunoprecipitation results showed the bindings between βarrestin2 and Arf6, Arf6 and EFA6A, and Arf6 and PLD1 Taken together, our findings strongly support the hypothesis that SS-induced SSTR2A endocytosis is mediated by the β-arrestin2- Arf6-EFA6A-PLD1 cascade in cell model system Therefore, we proposed a novel mechanism regarding SSTR2A endocytosis pathway in which via Gi/o-coupled SSTR2A, SS activates Gβγ, which triggers the GRK2 to phosphorylate SSTR2A, which is followed by recruitment of βarrestin2 β-Arrestin2 then forms the β-arrestin2-EFA6A complex in the cytosol and translocates it to the plasma membrane to activate Arf6 The activated Arf6 will stimulate PLD1 to catalyze the formation of phosphatidic acid (PA) from phosphatidyl choline (PC) PA then serves as a cofactor to activate phosphatidylinositol 4-phosphate-5-kinase (PIP5K) to increase PIP2 formation PIP2 ultimately recruits endocytic proteins such as adaptor protein (AP2) and clathrin to form vesicle-coat pits, resulting in SSTR endocytosis in β-cells iv     LIST OF ABBREVIATIONS AC: Adenylyl cyclase AP2: Adapter protein Arf6: ADP-ribosylation factor ARNO: Arf Nucleotide-binding site Opener Bax: Bcl-2–associated X protein β- Arr: β–arrestin cAMP: cyclic adenosine monophosphate cGMP: cyclic guanosine monophosphate CHO: Chinese hamster ovary DAG: Dicylglycerol DAPI: 4’,6-diamidino-2-phenylindole dilactate dn: dominant negative EDTA: Ethylenediaminetetraacetic acid EFA6: Guanine nucleotide exchange factor for Arf6 EFA6A: Guanine nucleotide exchange factor for Arf6 A EGFR: Epidermal growth factor receptor Erk: extracellular signal-regulated kinases FBS: Fetal bovine serum FLAG-tag: polypeptide protein tag GAPs: GTPase-activating proteins GEFs: Guanine nucleotide Exchange Factors GH: Growth hormone GIT1: G protein-coupled receptor interacting protein GPCRs: G protein-coupled receptors GRKs: G protein-coupled receptor kinases GRP: Cytohesin/general Receptor for Phosphoinosittides HA: Hemagglutinin HEK: Human embryonic kidney HRP: Horseradish peroxidase v     IP3: Inositol-1,4,5-triphosphate JNK: c-Jun-N-terminus kinase KRB: Krebs Ringer Buffer MAPK: Mitogen-activated protein kinases NHE: Na+/H+ exchanger PA: Phosphatidic acid PC: Phosphatidylcholine PH: Pleckstrin homology PKA: Protein kinase A PKC: Protein kinase C PI3: Phosphatidylinositol-3 PIP2: Phosphatidylinositol-4-5-biphosphate PIP3: Phosphatidylinositol 3,4,5-triphosphate PIP4Ks: Phosphatidylinositol 5-phosphate 4-kinases PIP5K: Phosphatidylinositol 4-phosphate-5-kinase PLA2: Phospholipase A2 PLC-β: Phospholipase C-β PLD: Phospholipase D PTP: Protein tyrosine phosphatase RINm5F: Rat insulinoma m5F RIPA: radioimmunoprecipitation assay SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis si-RNA: Small interfering RNA SS: Somatostatin SSTRs: Somatostatin receptors STAT3: Signal transducer and activator of transcription TSH: Thyroid-stimulating hormon     CHAPTER 1: GENERAL INTRODUCTION DISSERTATION ORGANIZATION This dissertation contains four chapters Chapter1: The general introduction includes a research objective, background information and literature review Chapter 2: Materials and Methods Chapter 3: Results: SS-induced SSTR2A endocytosis is mediated by β-arrestin2, Arf6, EFA6A and phospholipase D1 signaling pathway Chapter 4: General discussion and future research In addition, a list of references cited and acknowledgements are included RESEARCH OBJECTIVES Endocytosis or internalization of G protein-coupled receptors (GPCRs) is a process that is induced when extracellular agonists bind to surface receptors There are many signaling proteins participating in the endocytosis pathway of GPCRs such as G protein- coupled receptor kinases (GRKs), β-arrestins, ADP-ribosylation factor (Arf6), phospholipase D (PLD), phosphatidylinositol-4-5-biphosphate (PIP2), and adapter protein (AP2), (Doherty and McMahon, 2009; Houndolo et al., 2005; Sorkin and von Zastrow, 2009) Somatostatin (SS) receptors (SSTRs) undergo endocytosis following the agonist stimulation (Roosterman et al., 2008; Duran-Prado et al., 2007; Liu et al., 2005) However, the signaling pathway of SS-induced SSTR endocytosis is not well-understood In a previous study, we found that SS stimulated PLD activity and promoted PIP2 synthesis in β-cells (Cheng et al., 2005) We also demonstrated that in β-cells SS- stimulated PLD activity was mediated by a guanine nucleotide exchange factor for Arf6 A (EFA6A) (Grodnitzky et al., 2007) In addition, results from our preliminary study showed that upon SS stimulation SSTR2A underwent endocytosis Therefore, we hypothesized that β-arrestins, Arf6, EFA6A, and PLD participate in the SS-induced SSTR2A endocytosis signaling pathway The proposed pathway is shown in Scheme The objective of this dissertation is to     elucidate the role of four important signal proteins (β-arrestins, EFA6A, Arf6, and PLD) and their interrelationships in the SS-induced SSTR2A endocytosis pathway We used the rat insulinoma m5F (RINm5F) cell line transiently overexpressed with SSTR2A as a model We used radioligand binding techniques to demonstrate endocytosis To knockdown the signaling genes we either used small interfering RNA (si-RNA) techniques or overexpressed dominant negative (dn) constructs to generate mutant signaling proteins In addition, we used immunoprecipitation technique to investigate the interactions between signaling proteins     BACKGROUND AND LITERATURE REVIEW Somatostatin (SS) SS, a peptide hormone, was first discovered from the extraction of hypothalamus as a tetradecapeptide and inhibited secretion of growth hormone (Brazeau et al., 1973) Subsequently, SS was found to be secreted from many other tissues including δcells of pancreatic islets of Langerhans, nervous systems, and gastrointestinal mucosa, and in a small amount from the thyroid, adrenals, submandibular glands, kidneys, prostate, and placenta (Patel, 1999) It exists in two biologically active forms, SS-14 (14 amino acids) and SS-28 (28 amono acids) Both isoforms are derived from the proteolytic cleavage of the prosomatostatin (a 92-amino-acid protein), which is derived from a precursor of 116-amino-acid preprosomatostatin (Figure 1) SS has a potent inhibitory effect on the secretion of many hormones, including growth hormone (GH), thyroid-stimulating hormone (TSH), insulin and glucagon from the pancreas, and a number of gastrointestinal hormones such as gastrin, secretin, cholecystokinin, motilin, gastric inhibitory polypeptide and vasoactive intestinal peptide (Bloom and Polak, 1987; Kumar and Grant, 2010) Figure Somatostatin processing and amino acids order of SS-14 and SS-28 PreproSS: preprosomatostatin; proSS: prosomatostatin     SS receptors and their distribution There are five genes on different chromosomes encoding five different SS receptor subtypes (SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5) The name SSTR1-5 was based on the order of discoveries SSTR1 and SSTR2 were identified by Yamada et al (1992) Soon after that the rest of SSTRs (SSTR3, SSTR4, and SSTR5) were cloned and characterized (Patel et al., 1995, 1996; Patel, 1997) SSTRs have the sizes from 356 to 391 amino acids and have overall sharing of 39-57% sequence homology The most divergences are at the N- and C-terminal segments SSTR2 exists in two spliced variants named SSTR2A and SSTR2B; they are different at the C-terminus and are encoded on the same chromosome 17 SSTR2A is a physiologically active isoform between two of them (Barnett, 2003) SSTRs distribute in many cells/tissues, including brain, pituitary, islets, stomach, kidneys, liver, placenta, and lungs (Patel, 1999; Tulipano and Schulz, 2007; Olias et al., 2004) However, the distribution of SSTRs is not equal in all tissues There is usually more than one SSTR subtype in each cell or tissue SSTR1 is predominantly expressed in the brain and is also found in the pituitary, islets, and adrenals SSTR2 is predominantly expressed in many normal tissued including brain, vessels, nerve plexuses, adrenal medulla, spleen, kidneys, prostatic stroma, and pancreatic islets (Reubi et al., 2001; Liu et al., 2007; Reubi, 2003) The expression of SSTRs in neoplastic tissues is much higher in density than normal tissues in which SSTR2 is the most frequently expressed receptors of tumors (Reubi et al., 2001; Reubi, 2003; Liu et al., 2005) SSTR2 also has high affinity for SS analogs, therefore; SSTR2 is the most investigated SS receptor SSTR3 is highly expressed in the cerebellum, but less in the rest of the brain SSTR3 is also highly expressed in the spleen, kidneys, and the liver (Taniyama et al., 2005) SSTR4 is poorly expressed in the brain, but abundantly expressed in the heart and moderately expressed in the lungs and islets SSTR5 is prominent in the     pituitary, intestine, and islets, but poorly expressed in the brain (Taniyama et al., 2005) SS receptor signaling The actions of SS are mediated through SSTRs that are coupled to pertussis toxinsensitive Gi/o subfamily They are all GPCRs with seven transmembrane-spanning domains (TM1-7) (Patel, 1999; Lahlou et al., 2004), which are connected by three extracellular and three intracellular loops (Hu et al., 2010) G protein structures and actions GPCRs are activated by extracellular ligands, leading the receptors to interact with and activate G proteins G proteins are composed of three subunits (α, β, and γ) and are referred to heterotrimeric G proteins They are activated by diverse ligands, which vary from single photons through ions, odorants, amino acids, fatty acids, neurotransmitters, peptides/polypeptides, and proteins Upon activation, the GPCRs undergo conformational changes leading to the activation of G proteins by the exchange of GDP/GTP at Gα subunit The Gβγ dimer then dissociates from the Gα subunit Both moieties are active to stimulate downstream effectors and thereby initiate unique intracellular signaling responses (Tuteja, 2009) In humans, there are 21 Gα subunits encoded by 16 genes, Gβ subunits encoded by genes and 12 Gγ subunits encoded by 12 genes (Downes & Gautam, 1999) Based on sequence homology and functional similarities of α subunits, heterotrimeric G proteins can be grouped into four subfamilies: Gs stimulates adenylyl cyclase (AC); Gi/o inhibits AC and voltage-dependent Ca2+ channels, and acitvates K+ channels; Gq/11 activates phospholipase C-β (PLC-β); and G12 activates Rho, a 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research and the writing of this dissertation First and foremost, I thank my major Professor, Dr Walter H Hsu for he guidance, patience and support throughout this research and the writing of this dissertation He insights and words of encouragement have often inspired me and renewed my hopes for completing my graduate education Special thank to Vietnamese Government for the financial support for my PhD program I would also like to thank my committee members for their efforts and contributions to this work (Dr Hsu, Dr Martin, Dr Kimber, Dr Day, Dr Carlson, and Dr Kanthasamy) I want to thank to my professors, teachers and colleagues from my faculty and my department members at Nong Lam University, Vietnam for their helps and taking care of my job at the department during my absence I would additionally like to thank Dr Anantharam for his guidance about research during my internship at PKBiosciences I am thankful Cathy Martens, Nada Pavlovic, Kim Adams, Linda Erickson, and Bill Robertson and other BMS staffs for their helps during my time at BMS, ISU My family is the most important thing in my life I am thankful for their supports, encourage, and always with me in difficult times, especially my wife, Dung Pham and the present of my little daughter Lan-Chi Ngoc Vo push me to finish this dissertation This work is in memory of my father-in-law who always encouraged us, but couldn’t wait to see my graduation Finally, I am grateful to all of my friends here in Ames (especially Yih Ho’s family, Nikki’s family, Anh Chy’s family, Hai-Hong, Huy-Huong, Lam-Chi, Hariharam, Huajun Jin, Ahmed, Prince, Nalee, Michael, Mostafa) for their helps, discussing and sharing opinions about research and life in general [...]... al., 2007) SSTR2A < /b> is < /b> the most distributed receptor isoform of SSTR2 in human tissues and neuroendocrine tumors In addition, SSTR2A < /b> is < /b> the physiologically active isoform between SSTR2A < /b> and SSTR 2B (Barnett, 2003) In RINm5F cells, SSTR2 is < /b> the predominant SSTR isoform (Vo unpublished data) Therefore, in this dissertation I will focus my study on SS -induced < /b> endocytosis < /b> and signaling pathway of SSTR2A < /b> As... These findings elucidated the interactions between signaling proteins in the GPCR endocytosis < /b> In contrast, Cohen et al (2007) reported that Arf6 was activated less by ARNO than EFA6 in HeLa and COS-7 cells EFA6 family includes EFA6A, EFA 6B, EFA6C, and EFA6D EFA6A, EFA6C, and EFA6D are primary expressed in the brain and intestine, whereas EFA 6B is < /b> broadly expressed, but is < /b> not detected in the brain (Derrien... from our lab determined that EFA6A existed in β -cells (Grodnitzky et al., 2007) Because the findings from our lab indicate that EFA6A mediates SS -induced < /b> PLD activity through < /b> Arf6- PLD cascade (Grodnitzky et al., 2007), we hypothesize that EFA6A -Arf6 mediates SS -induced < /b> SSTR2A < /b> endocytosis < /b> in β -cells 19     13 Arf GTPase activating proteins (Arf GAPs) As discussed above, GEFs turn on the signaling for... Domain structures of Arf GAP subfamilies Abbreviations are: ALPS, Arf GAP1 lipid-packing sensor; Arf GAP, Arf GAP domain; ANK, ankyrin repeat; BAR, Bin/Amphiphysin/Rvs; BoCCS, binding of coatomer, cargo and SNARE; CALM, CALM binding domain; CB, clathrin-box; CC, coiled-coil; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; GRM, Glo3 regulatory motif; PBS, Paxillin binding... SS -induced < /b> PLD1 activation is < /b> mediated through < /b> EFA6A -Arf6 cascade (Grodnitzky et al., 2007) In RINm5F cells, we found both PLD1 and PLD2 (unpublished data) Therefore, we designed experiments to determine which PLD isoform was required for SS -induced < /b> SSTR2 endocytosis < /b> in RINm5F cells 15     10 PIP2 and endocytosis < /b> The lipid PIP2 accounts for only about 1% of total phospholipid in the plasma membrane,... publications regarding the involvement of Arfs in SSTRs endocytosis < /b> In this dissertation, I explored the role of Arf6 in the SS -induced < /b> endocytic signaling cascade for SSTR2A < /b> 12 GEFs and their role in endocytosis < /b> Arfs require GEFs to catalyze the exchange of GDP for GTP This group of proteins includes Gea/Gnom/GBF (Geal/2p, GBF1, Emb30/Gnom), Sec7/BIG (Sec7p, p200/BIG1/2, AL022604), ARNO/cytohesin/GRP... SS -induced < /b> SSTR2A < /b> endocytosis < /b> in β -cells is < /b> mediated by the β-arrestins -EFA6A -Arf6- PLD cascade Therefore, the objective of this dissertation is < /b> to elucidate the role of four important signal proteins (β-arrestins, EFA6A, Arf6, and PLD) and their interrelationships in the SS -induced < /b> SSTR2A < /b> endocytosis < /b> pathway I used RINm5F cells transiently overexpressed with SSTR2A < /b> as a model Cells were overexpressed... activation is < /b> required for receptor endocytosis < /b> and recycling and this endocytosis < /b> pathway is < /b> mediated by PLD2 (Rankovic et al., 2009) In angiotensin II type 1 receptor, Arf6 recruits AP2 and clathrin to receptor to activate the receptor endocytosis < /b> (Poupart et al., 2007) In addition, Arf6 is < /b> associated with clathrin-independent endocytosis < /b> in neuroblastoma cell (Kang et al., 2009) and beta 1 integrins (Dunphy... treatment of the cells with 1-butanol, a PLD inhibitor, inhibited endocytosis < /b> (Shen et al., 2001) These results confirmed the role of PLD in endocytosis < /b> PLD2, but not PLD1, has been shown to be required for endocytosis < /b> of µ-opioid receptor (Koch et al., 2004) and angiotensin II receptor (Du et al., 2004) In clonal β -cells HIT-T15, PLD1 is < /b> the only PLD isoform identified, which mediates SS -induced < /b> PIP2... Shenoy, 2005) In this dissertation, we used siRNAs as a molecular tool to knockdown β-arrestin 1 and β-arrestin 2 to explore which β-arrestin subtype is < /b> involved in SS -induced < /b> SSTR2A < /b> endocytosis < /b> in our cell model system 9 PLD and endocytosis < /b> PLD is < /b> a widely distributed enzyme that catalyzes the hydrolysis of phosphatidylcholine (PC) to form lipid second messenger phosphatidic acid (PA) (Jenkins and Frohman,