cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells

RcccivctlI 1 Febnrary 1691 The relariunxhip kt~tn

rwiwien ~nl’I~C chMP-tlrpcnthznl prutcirv kinnsc (cAMP~W) #nd l&and binding and intcrnalizrtiun by ~hr vasoprcnrin rsnut tV,4ypfr~plarol LLGPK, rcn~l epirhcli#l eiclirwascxrmlnrd. U~CW~AMP+K nctivatian rhr#upb I h trc#Imcnt with thetAMP rnrlsga 8.brumu~cAh”lP (WrrAI. n rxirkcd rcducrienin V,-rrscplarxrcircly ~tutenumberanctintcm~lixrrtinn in LLGPK, c&r WIS cfWcd. In ecllr treated for 17 h with WA and hence &wn+qul~tctdfor eAMBPK,the Vaarcecplarnumber v&x narmrl but intcrn~lhutitm wux markedly rcdurcd. Cella or the LLC.PKImrrlunlFILPJI. which~ONCWIX nheul 10% parent&l cAMIWC catalytic subunit uerivity, rxhibitrd lower V,~r~cprar w~cndySIPI~ number nnd inlern&sWm in conrptwisun lu untrewd LLC-PK, strlla. A nqntivdeurrcleriorn WHX thusevident bd~ween chMP+tC ttetivulion and V,-rcecptar number. mid intcmatliwtirln. Fhelrpharyhltian by eAMPJ% may elka ligrnd4ndcpcndcnr remaval al’ reecpmrrrom ths pluamu membrunt,

Dawn-rcgulWm;

1,

Pen#l cpithrliul ecll: VnnoprcsSin

V,-rcccptstin~crn~~liz~tian; cAMP.dcpendcnr

INTRODUCTION

Phosphorylation is a universal regulatory mechanism of cellular processes including growth, differentiation and transformation, In the adenylate eycIasc (AC) systern, the CAMP-dependent protein kinase (CAMP-PK) is activated by a concerted series of events initiated by the binding of hormone to receptor at the external surface of the plasma membrane [ 1,2], CAMP production by AC leads to the elevation of intracellular CAMP levels, and dissociation of the CAMP-PK holoenzyme complex to release the active catalytic (C-) subunit [3,4]. A variety of cellular proteins serve as substrates for phosphorylation by the kinase, resulting in the modulation of a number of metabolic pathways and gene regulation [1,2]. Down-regulation or desensitization begins at the level of the receptor [5-71, but occurs at all stages of the above [2,5,8], including CAMP-PK itself [9,10]. The cAMP-PK [5-7,11,12] as well as the ,&adrenergic

receptor kinase [13,14] are capable of phosphorylating the ,&adrenergic receptor, thereby regulating its transducing capacity and/or endocytosis, This study examines the effect of in vivo activation of D.A. Jans, c/o Dr. F. Fahrenholz, Max Planck Institut fiir Biophysik, Kennedyallce 70, D-6000 Frankfurt am Main 70, Germany. Fax: (49) (69) 630 3423

Correspondetm

oddms:

CAMP-PK on the vasoprcssin &-type receptor of LLCPK, renal cpithcliai cells [lS] which possess distinct receptors for vasoprcssin and calciconin, both of which activate AC [16,17]. In addition to agents elevating intraccllular CAMP, phorbol csccrs stimulate LLC-PKI cells lo produce urokinase-type plasminogen activator by a CAMP-independent Ca’+Jphospholipid-dependent protein kinase (PK-C) -mediated pathway [18,19]. Here we examine cells of the LLC-PKI cell line, and those of the FIB4 mutant, which possesses normal amounts of CAMP-PK C-subunit, but only aboGt 10% wild-type activity f 18,20), Treatments activating CAMP-PK or PKC to differing extents were examined for their effects on V2-receptor binding. We demonstrate a nega.tive correlation receptor

Published

by Elsevier Science Publishers 5. V.

between CAMP-PK activation number and internalization.

and both VZ-

2. MATERIALS AND METHODS 2 I I t Mareriols

5’-[y-3LPJATP and [~WJArgs-vasopressin (AVP) were from Amcrsham, and phosphocellulose paper (P-81) from Whatman. All Other materials were from previously described sources [17,21]. 2.2. Cell culrure The LLC-PKI pig kidney epithelial cell line [IS] and the FIB4 mutant [18,20) were cultured as described previously [17]. 2,3. Enzyme

CAMP-PK, CAMP-dependent protein kinase or ATP I protein phosphotransferase (EC 2.7.1,37); CAMP, adenosine 3 ‘,5 ‘monophosphate; BrcA, 8-bromo-CAMP; AC, adcnylate cyclase or ATP pyrophosphatelyase (cyclicizing, EC 4.6.1.1); AVP, Arg*vasoprcssin; IBMX, 1-isobutyl-3-methylxanthine Abbreviuriom:

protein kinunc

us.wj~s

Extracts for the assay of CAMP-dependent protein kinasc (CAMPPK) catalytic activity were prepared and assayed using Kemptide (LR-R-A-S-A-G) as a substrate 117,211. The CAMP-PK activity ratio expresses rhe C-subunit activity present in cell extracts (assayed in the absence of CAMP) relative to the total stimulatable activity (assayed

3.

RESULTS

WC initially modified our binding assay for LLGPK, ccllt in suspension [I71 to cplantitntc the relative contribution of receptor internalization/recycling etc. to total maximal Y&receptor bi.nding activity (section 2). Half-maximal binding at 30eC and 4’C was achieved at 2.2 f 0,l min (Fig- 1, squares) and lg.6 f 1.4 min (Fig. 1, closed circles) respectively (mean & SEM for n > 3). Lignnd internalization, estimated using a pW 3 treatment subsequent to binding, was half-maximal at 6.9 f 1.6 min at 30°C (Fig. 1, triangles), No intcrnalizntion was observed at 4’C (Fig. 1, empty circles). Maximal specific binding at 4OC (64 fmol/lO” cells)

0

15 30

60

90

120

time tmin) Fig. 1. Time course OF [‘HIAVP

specific binding and internalization in EDTA-suspended LLC-PKI cells. Total specific binding at 30°C (S--e) or 4°C (S--a), or internalization at 30°C (F--V) or 4OC @----O) was determined as described in section 2. Results are the means From a single typical experiment For which the SEM was less than 11% the value of the mean. Maximal binding was achieved at 40.0 -L 3.4 and 10.8 + 2.1 min (mean f SEM, n > 3) at 4°C and 3O”C, respectively; internalization was maximal at 15.7 2 3.3 min.

268

represents the number of binding sites at steady state (no conrribucion of internalization or rceeptor cycling/recycling); and accounted for about 53aloof total maximal binding at WC (120 fmol/106 eclls). Internalization at 30°C (40 fmol/lO” cells) accounreci for a further 33%, which is comparable to that observed in other systems (e.g, [28]). Short trypsin treatment (0.25ol0, 5 min, 4OC) subsequent to binding was not as effective as pH 3 treatment in removing non-internalized ligand, since 29”/o of ligand bound at 4*C remained cell-associated (Table I), Cells pretreated at pH 3 prior to binding showed no reduction of either maximal specific [3H]AVP binding nor internalization at 4°C or 30°C (Table I), indicating that pH 3 treatment did not irreversibly denature the V,-receptor. In contrast, short trypsin pretreatment reduced maximal binding by 33% or 56% at 4°C or 3O”C, respectively. NaN3, which inhibits energydependent processes such as rcccptor endocytosis, did not affect the number of AVP-binding sites (4’C binding), but reduced internalization by 5 1% compared to untreated cells (Table I). 3.1. L3H_/4W bindkg and ~~ter~Qiiz~~i~nin LLC-PKI cells treated with ugem elevating intracelluluc CAMP Ievels LLC-PK! cells were pretreated with the CAMP analogue 8-bromo-CAMP (BrcA) for either 1 h or 17 h, and then CAMP-PK activation (Table II) and maxima1 specific [‘H]AVP binding and internalization were determined (Fig. 2). 1 h treatment (CAMP-PK activity ratio of 0.42) resulted in a marked reduction of both AVP internalization (54% reduced compared to untreated cells) and binding at 4°C (35070decreased). 17 h treatment (conditions of down-regulated CAMP-PK; activity ratio of 0.24) also induced a reduction in internalization (5 1070reduced compared to untreated con-

rrol). Reduced AVP inrcrncsliaatian did nor ~ppcsr NJ rcsulr from an altered Vz-receptor affinity for ligand (nor shown). Results were compared to cells pretreated for 1 h or 48 h with 3 x IO”” M phorbol-tnyristate acetate (PMA), frcatmcnts which induce activation and down-regulation

-I

b°C LLC-PK, r’

1

FIB4

1

LLC-PK,

FIB4

1

T

respectively of PM-C [18,19,29], Compared to untreated cells, PMA-trcaccd cells exhibited slightly increased internalization at 30*Cand binding at 4’C. Total binding at 30°C was rsxentially comparable for all oft hc variously treated and untreated cells, with the possible exception of PK-C-down-regulated cells, which showed 19% higher total binding. The data for LLC-PK, cells under the above various conditions (from Table II and Fig. 2) revealed a negative correlation (v = -0.92, x = 65.8, y = -96.7, /I = S) bcfween the cAMP-PM activity ratio and the number of Vz-receptors at steady state (binding at 4OC), A negative correlation (I’= -0.89, x = 47. I, y F -69.6, 11= 5) was also observed for CAMP-PK activity ratio and AVP internalization. 3.2. flYJAW CAMP-PK

Fig. 2. Maximal specific [“H]AVP bindinE and internalization in cc!is of the LLC-PKI and CAMP-PK mutant FIW cell lines. Total specific binding ( q) and internalization (a) were determined after 30 and 60 min at 30°C and 4OC, respectively, on cells pretreated as indicated. The results shown are the means with SEM shown for more than 3 experiments performed in duplicate.

binding and internulization C-subunit r?zutm FIB4

in

the

The FIB4 mutant [20] was similarly analyzed for AVP binding and internalization. FIB4 cells, with or without BrcA pretreatment, showed total binding at 30°C essentially comparable to that of LLC-PKI ceils (Fig. 2). The extent of CAMP-PK activation (CAMP-PK activity ratio) upon BrcA treat,ment (I h or 17 h) was also comparable for both cell lines (Table II), although the absolute CAMP-PK activities were largely different due to the C-subunit mutation of FIB4. However, maximal specific [3H]AVP internalization was markedly lower (more than 40% reduced), as was steady state binding activity (4OC) (50% reduced) compared to untreated LLC-PK, cells. FIB4 cells thus largely resembled BrcA-treated (down-regulated) LLC-PKI cells in 269

their [rH]AVP rready IWF: binding and lnre~nali%~tion (Fig. 2).

cellular cAMP levels have an effect on AVP-binding, LLC-PKI cells were prerrerred with either forxkolin (AC ncrivarot) or salmon calcitonin for 17 h snd eampared to Brch-treated or uncrrated fells for [“WIAVP binding (Fig, 3). All n,goniet pretrcatmcntd reduecd [‘I-I] AVP in~ernali2a~iol~ (Fig. S), indicating tkar agcntrc ocher than BreA which bring about cAMP-PK do&%regulation [IO] also effect a reduction in V~~~pfor inrernaliration. 4. IXSCUSSION The results here suggest a role fur the CAMP-PK in regulating liynnd binding and inrcrnalization by the VZreceptor of LLC-PKt cells. Trcatmcnlf resulting in rhe stimulation and subsequent down-regulation of CAMPPK markedly reduced the steady state V~~cp~or number and internalization. Interestingly, the CAMP-PK Csubunit mutant FIB4 also showed a low steady srare receptor number and internalization, Activation of the CAMP-PK by BrcA (and probably also by vasopressin itself) appears to effect endocytosis of plasma mcmbranc receptors even though they arc not occupied by ligand, and concomitant reduction in ligand-dcpcndcnt internalization, presumably mediated by phosphorylation, That hcterologous hormone (calcitonin)or

I

1

’

I

,

’

L

1

0

15

30 Time

1

1

,

1

*

25

60

(min)

Fig. 3. Influenceof agents elevating intracellular CAMP on I’H]AVP internalization in LLC-PKI cells. The timecourse of internalization at 30°C was followed in LLC-PKI cells which had been pretreated for 17 h without (o-d) or with 1 mM BrcA (V--V), IO ~4ctMforskolin ( D---D), or 30 nM salmon calcitonin (gd), Results are the means from a single typical experiment for which the SEM was less than 12% the value of the mean.

290

O\her

kinarts

may alsa hwc an int’luencs on Ww

reprsr function. P&C rhc @adrener$ic ry~rn marked eft”rcr of PMA an elevk\rcd rather than number (9x Fig. 21, In ~umour necro%ia thctor tar down-regulation in reduction in the number

pl~y’~ a rale in dcPenrririxarion in [5,31], bur we obscrvrid no an Va.-inrernalizution here, and rcducrd arcady state VZ-receptor the eaxe a$ xhc recepr0r4 for the and EGF, PMA ind~iecs rccepthe absence af lipand, due to a

of plasmn membrane reecptorr [32]. lnrcrcstingly, 0~i.u pnrallcls rhc effceta of’ cAMI> agonists on the Vp-receptar. Further examination of the various complex fecdbeck mcchsnisms usin in viro systems, together with murant% affected in specific COW ponents of signal lransdutxion, sllould assist in elucidating the praccnrcs reyulating receptor function,

REFERENCES

11) Lohmnnn,

S.M,

mrtl \Qtltcr, U. (19X4) Adv. Cyclic Nuclcnt. Rer. I#, 63-117, PI Steinberg, R.A. (19X3) Binrhcrn. Actions tlorm. I I, 25-65. I31 Flockhart, D.A, ihntl Corbin, J,D. (1982) CRC Crit. Rev. Biochcm. I?, 133-186. I41 Dechrcl, P.J,, Bravo, J.A. nnd Krcbs, E.G, (1977) .I, Bial. Chcrn. 252, 2691-2697, R.J. ISI Denovic, J.L., Bouvicr, M,, Cnron, M,G. and Lcfkowitz, (19XH) Annu. Rev. Cell Biol. 4, 4O5-42R. R.J. (1967) Nature 317, 124-129. I61 Sibley, D,R. and Lcfkawitz. R.J. I71 Sibley, D.R.. Strasscr, R,H., Caron, M.G. and Lcfkowitz, (1986) Cell 48, 913-918, PI Jans, D.A. and Hemmings, B.A. (1988) Adv. Second Mesrcnycr Phasphoprotein Res, 21, 109-121. E., Taubcr.Finkclstcin, M., Schmceda, H. and PI Alhnnaty. Shnltiel, S. (1985) Curr. Top, Cell. Regulatio:l 27, 267-27X. 1101 Hemmings, i3.A. (1986) FEDS Lett, 196, 196-200. Pctcrs, J.R., Nambi, P.. Caron, M.G. and II 11 Sibley, D,R., Lcfkowitz, R,J, (1984) J. Biol. Chcm, 259, 9742-9749. iI21 Benovic, J.L,, Pike. L.J., Ccrionc, R.A., Staniszewski, C., Yoshimasa, T., Codina, J., Caron, M,G, and Lcfkowitz, R.J. (198s) J. Biol. Chem, 260, 7094-7101. H., Mayor, F., Cotccchia, iI31 Benovic, J.L., Rcgan, J,W., Mat&, S., Lceb-Lundberg, L,M.F,, Caron, M.G. and Lefkowitr, R.J. (1987) J. Biol. Chcm. 262, 17251-17253, [I41 Benovic, J,L., Kuhn, H,, Wcyand, I., Codina, J ,, Caron, M,G. and Lefkowitz, R.J. (1987) Proc. Natl. Acad. Sci. USA 84, X879-8882. (151 Hull, R.N,, Cherry, W.R. and Weaver, GW. (1976) In vitro 12, 670-677. [16] Daycr, J.-M., Vassal& J.-A., Bobbit, J.L., Hull, R.N., Reich, E.R. and Krane, S.H. (1981) .I. Cell Biol. 91, 195-200. [17] Jans, D.A., Resink, T.J., Wilson, E.R., Reich, E. and Hemmings, B.A, (1986) Eur, J. Biochem. 160, 407-412.

Pro!. Phosphorylfltion

Volume 381, number I .2

12s) Br%tlltwtl, W.M.

(19761 Awl,

tliaehem.

7’3, 2JI4fS.

291