Essential Role for G Protein-coupled Receptor Endocytosis in the Activation of Mitogen-activated Protein Kinase

The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and β-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of β-arrestin or dynamin, β2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and β-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade. The classical paradigm for G protein-coupled receptor (GPCR) signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G proteins at the plasma membrane and the subsequent generation, by membrane-localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G protein-coupled receptor kinase (GRK)- and β-arrestin-mediated receptor uncoupling and internalization. Here we show that these paradigms are inadequate to account for GPCR-mediated, Ras-dependent activation of the mitogen-activated protein (MAP) kinases Erk1 and -2. In HEK293 cells expressing dominant suppressor mutants of β-arrestin or dynamin, β2-adrenergic receptor-mediated activation of MAP kinase is inhibited. The inhibitors of receptor internalization specifically blocked Raf-mediated activation of MEK. Plasma membrane-delimited steps in the GPCR-mediated activation of the MAP kinase pathway, such as tyrosine phosphorylation of Shc and Raf kinase activation by Ras, are unaffected by inhibitors of receptor internalization. Thus, GRKs and β-arrestins, which uncouple GPCRs and target them for internalization, function as essential elements in the GPCR-mediated MAP kinase signaling cascade. Stimulation of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; MAP, mitogen-activated protein β2-AR. β2-adrenergic receptor; RTK, receptor tyrosine kinase; LPA, lysophosphatidic acid; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; ISO, isoproterenol; PMA, phorbol 12-myristate 13-acetate. facilitates the exchange of bound GDP for GTP on heterotrimeric G proteins, resulting in dissociation of the G protein into active Gα-GTP and Gβγ subunits. The interaction of Gα-GTP and Gβγ subunits with effectors initiates and accounts for the known signaling events mediated by GPCRs. Exposure of GPCRs to an agonist often results in rapid attenuation of receptor responsiveness, a process termed desensitization. Signal termination is initiated by phosphorylation of agonist-occupied receptors, mediated by the G protein-coupled receptor kinase (GRK) family (1Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1088) Google Scholar, 2Inglese J. Freedman N.J. Koch W. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 23735-23738Abstract Full Text PDF PubMed Google Scholar, 3Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (474) Google Scholar). The GRK-mediated phosphorylation of activated GPCRs promotes binding of members of a family of cytosolic proteins, β-arrestins, to the receptor (4Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 5Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). Binding of β-arrestins to phosphorylated receptors serves two functions. First, it uncouples the receptor from its cognate G protein and thus leads to diminished receptor signaling (4Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 5Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar). Second, it initiates the process of receptor internalization (also termed sequestration) by targeting the receptor to clathrin-coated pits (6Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar, 7Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Pen R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar). G protein-coupled receptors and receptor tyrosine kinases (RTKs) stimulate mitogenesis in part via mitogen-activated protein (MAP) kinase cascades. The mechanism of activation of MAP kinase signaling pathways by GPCRs is poorly understood, although it is becoming evident that signal transduction by certain GPCRs utilizes many of the same intermediates as those activated by RTKs (see Reaction 1). GPCR→Gβγ→Tyr kinase→Shc→Grb2­mSos→Ras→Raf→MEK→MAPKReaction 1 In fibroblasts, endogenous lysophosphatidic acid (LPA) and β2-adrenergic (β2-AR) receptors, acting via unknown effectors of Gβγ subunits, stimulate tyrosine phosphorylation of plasma membrane-associated proteins to create tyrosine phosphoprotein scaffolds (8Rao G.N. Delafontaine P. Runge M.S. J. Biol. Chem. 1995; 270: 27871-27875Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 9Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 10van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (526) Google Scholar, 11Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar, 12Luttrell L.M. DellaRocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Receptor activation coincides with an increase in tyrosine phosphorylation of the adaptor protein Shc (10van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (526) Google Scholar, 11Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar, 12Luttrell L.M. DellaRocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), and recruitment of Ras guanine exchange factors, such as the Grb2-mSos complex, to the plasma membrane. Recruitment of mSos facilitates Ras GDP/GTP exchange leading to recruitment of Raf (MAP kinase kinase kinase) into complex with activated Ras. Subsequent signal transduction involves sequential phosphorylation of the MEK (MAP kinase kinase) and MAP kinases (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-713Crossref PubMed Scopus (390) Google Scholar). Here we demonstrate that, unlike classical GPCR-mediated activation of adenylate cyclase or phospholipase C which occurs entirely within the plasma membrane, MAP kinase activation involves GPCR sequestration. Human embryonic kidney (HEK) 293 cells, maintained in minimum essential medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin were transiently transfected using calcium phosphate coprecipitation (14Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Cells were starved overnight in medium containing 10 mm Hepes (pH 7.4) and 0.1% (v/v) bovine serum albumin prior to agonist stimulation. All assays were performed 48 h after transfection. Transient expression of β-arrestin1, β-arrestin1 V53D, dynamin, and dynamin K44A transfected plasmids were verified by immunoblotting of whole cell lysates using commercially available antibodies. The β2-AR sequestration was determined by immunofluorescence flow cytometry (15Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar). Cells expressing epitope-tagged β2-AR at 300–400 fmol/mg of whole cell protein were exposed to 10 μm isoproterenol for 30 min at 37 °C prior to addition of antibodies. Sequestration is defined as the fraction of total cell surface receptors which are removed from the plasma membrane (and thus are not accessible to antibodies added to the cells) following agonist treatment. Cells were metabolically labeled with 1 μCi of 3Hadenine/ml, washed in PBS, and incubated with 1 mm isobutylmethylxanthine for 25 min at 37 °C. Agonist was added for 5 min followed by the addition of 1 ml of stop solution (0.1 mm cAMP, 4 nCi of 14CcAMP/ml, 2.5% perchloric acid). Cell lysates were neutralized with KOH, and total 3HcAMP was assayed by anion exchange chromatography (14Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Cells were metabolically labeled for 16–18 h with 2 μCi of 3Hinositol/ml and washed in PBS containing 20 mm LiCl alone (basal) or with agonist for 5 min at 37 °C. Reactions were terminated by the addition of an equal volume of 0.8 m perchloric acid, and total inositol phosphates were assayed by anion exchange chromatography (14Oppermann M. Freedman N.J. Alexander R.W. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Agonist-treated cells were lysed by direct addition of Laemmli sample buffer. Aliquots were resolved by SDS-PAGE, and phosphorylated MAP kinases on nitrocellulose filters were detected using a phosphospecific MAP kinase IgG (Promega). Bands corresponding to MAP kinase were visualized with enzyme-linked chemiluminescence (ECL; Amersham Corp.) and quantitated by scanning laser densitometry. Agonist-treated HEK293 cells were lysed in RIPA buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 10 mm NaF, 10 mmsodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate) and clarified by centrifugation. Shc proteins were immunoprecipitated using rabbit polyclonal anti-Shc antibodies (Transduction Laboratories) and resolved by SDS-PAGE. Phosphorylated Shc proteins on nitrocellulose were detected using anti-phosphotyrosine antibodies (RC20H, Transduction Laboratories), visualized with ECL, and quantitated by scanning laser densitometry. Lysates were prepared in RIPA buffer from cells treated with agonists for 5 min at 37 °C. Raf-1 was immunoprecipitated with 0.5 μg of anti-Raf-1 polyclonal antibody (C-12, Santa Cruz Biotechnology). Immunocomplexes were washed with cold RIPA, wash buffer (137 mm NaCl, 20 mm Tris, pH 7.4, 2 mm EDTA, 1.5 mm MgCl2, 1 mm dithiothreitol, 1 mmNa3VO4, 10% glycerol, 1% Nonidet P-40), and kinase buffer (75 mm NaCl, 20 mm Tris, pH 7.4, 1 mm EDTA, 1.5 mm MgCl2, 1 mm dithiothreitol, 1 mmNa3VO4, 30 μm ATP). Raf-1 kinase activity was determined by incubating the resuspended immune complexes in kinase buffer containing 10 μCi of 32PATP and 0.5 μg of MEK at ambient temperature for 15 min. Reactions were terminated by the addition of SDS sample buffer, and the phosphorylated substrate bands were resolved by SDS-PAGE and quantitated by phosphorimaging. Cells stimulated with or without 10 μm isoproterenol were washed with ice-cold PBS, scraped into 5% (w/v) sucrose in buffer A (10 mm Tris, pH 7.4, 1 mm EDTA), and disrupted by Dounce homogenization (16Graham J.M. Dealtry G.B. Rickwood D. Cell Biology Labfax. Academic Press, New York1992Google Scholar). Nuclei and cell debris were removed by centrifugation at 500 × g for 10 min. The supernatant was loaded on a sucrose cushion (4 ml of 35% sucrose in buffer A) and centrifuged at 150,000 × g for 90 min at 4 °C. The 35% (vesicle) sucrose interface fractions were collected, diluted with buffer A, and pelleted. The pellets were resuspended in SDS sample buffer; 25 μg for each protein sample was analyzed. The presence of clathrin and Raf was detected by protein immunoblotting. Clathrin was detected using a 1:500 dilution of a monoclonal anti-clathrin IgM (ICN), and Raf-1 was detected using a 1:1000 dilution of a rabbit polyclonal anti-Raf-1 IgG (Santa Cruz Biotechnology). Immune complexes on nitrocellulose were detected using the appropriate horseradish peroxidase-conjugated secondary antibody and visualized by ECL. Binding assays were performed exactly as described (17Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). To determine the role of GPCR internalization in signal transduction we employed dominant suppressor mutants of β-arrestin1 and dynamin. Dominant suppressor β-arrestin1 (V53D) prevents GPCR targeting to clathrin-coated pits, while the dominant suppressor form of dynamin (K44A) inhibits fission of the budding vesicle from the plasma membrane (18McClure S.J. Robinson P.J. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (75) Google Scholar). Fig. 1 Adepicts the effects of wild type and mutant β-arrestin1 and dynamin expression on β2-AR internalization. Whereas overexpression of the wild type β-arrestin1 protein increases isoproterenol-mediated internalization of the β2-adrenergic receptor (β2-AR) modestly, overexpression of wild type dynamin has no effect. In cells overexpressing the dominant suppressor forms of these proteins (β-arrestin1 V53D or dynamin K44A) a dramatic decrease in agonist-mediated internalization of the β2-AR is observed, in agreement with previous results (6Ferguson S.S.G. Downey III, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar, 19Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). The β-arrestin1 V53D mutant inhibits sequestration of the receptor by 50%, and the dynamin K44A mutant inhibits sequestration by 70%. As shown in Fig. 1 B, cells expressing β-arrestin1 V53D or dynamin K44A exhibit normal β2-AR coupling efficiency to Gs, as measured by the accumulation of intracellular cAMP. Similarly, coupling of the LPA receptor to Gi, as measured by the accumulation of intracellular inositol phosphates, was unaffected by expression of β-arrestin1 V53D or dynamin K44A proteins (Fig. 1 C). Thus, neither inhibitor of receptor sequestration significantly impaired classical receptor-G protein-effector-mediated generation of soluble second messengers. Stimulation of the endogenous receptors for lysophosphatidic acid (LPA) and isoproterenol (ISO; β2-AR) in HEK293 cells induces a 6- to 8-fold increase in phosphorylated MAP kinase (Erk1/2) levels (Fig. 2 A). Activation of MAP kinases by the β2-AR in HEK293 cells, like in Cos-7 cells (20Crespo P. Cachero T.G. Xu N. Gutkind J.S. J. Biol. Chem. 1995; 270: 25259-25265Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), is c-Src- and Ras-dependent (21Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1077) Google Scholar). Phosphorylation of MAP kinase reflects the enzymatic activation of MEK (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-713Crossref PubMed Scopus (390) Google Scholar). Expression of β-arrestin1 V53D and dynamin K44A mutants impaired the ability of these receptors to activate MAP kinase (Fig. 2 A). β-Arrestin1 V53D inhibited LPA- and ISO-mediated phosphorylation of MAP kinase by 56% and 63%, respectively. Similarly, the dynamin K44A protein inhibited LPA- and ISO-stimulated phosphorylation of MAP kinase by 60% and 55%, respectively. Other known inhibitors of GPCR sequestration, concanavalin A, monodansylcadavarine, and low temperature (22Waldo G.L. Northup J.K. Perkins J.P. Harden T.K. J. Biol. Chem. 1983; 258: 13900-13908Abstract Full Text PDF PubMed Google Scholar, 23Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 24Hoffman J.F. Linderman J.J. Omann G.M. J. Biol. Chem. 1996; 271: 18394-18404Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), all inhibited LPA- and isoproterenol-stimulated MAP kinase phosphorylation by approximately 70% (data not shown). In contrast, as shown in Fig. 2 B, phorbol ester (PMA)-stimulated MAP kinase phosphorylation in HEK293 cells, which is not receptor-mediated, is not affected by the presence of β-arrestin1 V53D or dynamin K44A proteins. Together, these data demonstrate that inhibition of receptor sequestration attenuates activation of MAP kinase without affecting early, plasma membrane-delimited, signaling events such as receptor coupling to G proteins. Signal transduction between receptors on the plasma membrane and MAP kinase in the cytosol requires a series of events leading first to the assembly of an activated Ras-Raf complex on the membrane, followed by initiation of the cytosolic MEK-MAP kinase cascade. To determine the point in the signaling cascade at which receptor internalization is required, we assayed the effects of β-arrestin1 V53D and dynamin K44A on two intermediate steps in the pathway: tyrosine phosphorylation of the Shc adaptor protein and activation of the Raf kinase. Activation of protein-tyrosine kinases by GPCRs and RTKs is obligatory for signal transduction to MAP kinases (8–13; see Reaction 1). Coincident with the increase in protein-tyrosine kinase activity is the tyrosine phosphorylation of the adaptor protein Shc, thought to play an important role in the nucleation and assembly of the multiprotein, Ras activation complex (11Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar, 12Luttrell L.M. DellaRocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). In HEK293 cells, stimulation with LPA or isoproterenol results in an increase of 4.2- or 3-fold, respectively, in tyrosine phosphorylation of Shc relative to unstimulated cells (Fig.3 A). Expression of neither β-arrestin1 V53D nor dynamin K44A proteins had any effect on LPA- or ISO-stimulated Shc phosphorylation (Fig. 3 A). Stimulation of the endogenous receptors for LPA or isoproterenol increases Raf-1 enzymatic activity 2-fold relative to unstimulated cells, in agreement with results reported recently in rat hepatocytes (25Spector M.S. Auer K.L. Jarvis W.D. Ishac E.J. Gao B. Kunos G. Dent P. Mol. Cell. Biol. 1997; 17: 3556-3565Crossref PubMed Scopus (98) Google Scholar). In cells expressing β-arrestin1 V53D or dynamin K44A mutant proteins Raf-1 activity was similar to that in wild type cells following stimulation of these endogenous receptors (Fig. 3 B). Thus, the initial steps in the GPCR-mediated MAP kinase cascade, including tyrosine phosphorylation of adaptors and activation of Raf, are unaffected by inhibitors of receptor sequestration. These data demonstrate that the process of vesicle-mediated endocytosis is required for mitogenic signaling initiated by G protein-coupled receptors. Inhibition of endocytosis blocks phosphorylation of MAP kinase, but does not affect plasma membrane-delimited processes such as receptor coupling to G proteins, tyrosine phosphorylation of Shc, or Raf activation. Rather, inhibition of endocytosis impairs signal transduction between activated Ras-bound Raf and the cytosolic MEK kinase. Regulation of Raf kinase activity has been shown to be complex; dependent, in part, upon its translocation to the plasma membrane and binding to Ras and 14-3-3 proteins, serine and tyrosine phosphorylation, and oligomerization (26Avruch J. Zhang X. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (542) Google Scholar, 27Morrison D.K. Cell Biol. 1997; 9: PubMed Scopus Google Scholar). data an mechanism for the of targeting the activated form of the to an intracellular its β-arrestin V53D and dynamin K44A effects of Raf, does inhibition of β-arrestin the mitogenic signaling cascade initiated by the A is that the receptor part of a signaling complex, at the plasma membrane, which not the but intermediates in the to and including Stimulation of HEK293 cells with isoproterenol results in the internalization of of the receptor into a (17Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). In agonist induces recruitment of soluble clathrin the plasma membrane it forms that a membrane vesicle (18McClure S.J. Robinson P.J. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (75) Google Scholar). fission of the vesicle from the plasma membrane, clathrin from the vesicle into the cytosolic the agonist-mediated translocation of β2-AR clathrin and Raf proteins to the These data the that agonist the assembly of a signaling complex, including Raf, which is by the clathrin-coated vesicle These not the that Raf internalization, of is required for signal A of and mitogenic signal transduction pathways to MAP receptor many of the same intermediates in signaling cascade (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-713Crossref PubMed Scopus (390) Google Scholar). inhibition of internalization was to the phosphorylation of the A. Science. 1997; PubMed Scopus Google Scholar). in cells, receptor-mediated activation of phospholipase is but phosphorylation of MAP kinase is impaired C. Science. 1996; PubMed Scopus Google Scholar). these data demonstrate between RTKs and GPCRs in mitogenic signal a for receptor internalization via clathrin-coated T. for the dynamin and P. Dent for D. for data prior to A. for with the Raf and J. W. and D. Luttrell for and of the M. and D. for