The EMBO Journal vol.9 no.9 pp.2693 - 2699, 1990

Lateral mobility of the phospholipase C-activatingvasopressin V1-type receptor in A7r5 smooth musclecells: a comparison with the adenylate cyclase-coupledV2-receptor

David A.Jans, Reiner Peters andFalk Fahrenholz

Max-Planck-Institut fur Biophysik, Kennedy Allee 70, D-6000Frankfurt am Main 70, FRG

Communicated by B.Hamprecht

The present work examines lateral mobility of thevasopressin VI-type receptor, representing the firstdetermination of lateral mobility of a hormone receptorcoupled to phospholipase C activation. The VI-receptorof A7r5 smooth muscle cells was characterized for [Arg8]vasopressin (AVP) binding properties and affinity for thefluorescent vasopressin analogue 1-deamino[8-lysine(M-tetramethylrhodamylaniinothiocarbonyl)] vasopressin(TR-LVP). TR-LVP was biologically active in A7r5 cells,inducing inositol 1,4,5-trisphosphate turnover in similarfashion to AVP. TR-LVP was used to specifically labelthe VI-receptor of living A7r5 cells, and lateral mobilityof the Vl-receptor was measured using the technique offluorescence microphotolysis. The apparent lateraldiffusion coefficient (D) at 37°C was 5.1 x 10-10 cm2/s,failing to 2.9 x 10-10 cm2/s at 13°C. These D values arehigher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK, renalepithelial cells analysed with the same fluorescent ligand.In contrast to the V2-receptor, no marked temperaturedependence was observed for the VI-receptor mobilefraction (f). From 37°C to 13°C, f was relatively low(between 0.4 and 0.5) consistent with Vl-receptorimmobilization through internalization, which is rapideven at room temperature in A7r5 cells. These differencesbetween V1- and V2-receptor lateral mobility arediscussed in terms of the implications for their respectivesignal transduction systems.Key words: lateral mobility/photobleaching/smooth musclecell line/vasopressin VI-receptor

Introduction

The antidiuretic hormone vasopressin is a major regulatorof blood volume and blood pressure. It acts through bindingto specific membrane receptors, the VI-vascular andV2-renal types (Jard, 1983), which stimulate thephospholipase C/inositol 1,4,5-trisphosphate (Berridge andIrvine, 1984; Siess et al., 1986) and adenylate cyclase/cAMPsecond messenger pathways, respectively. Activation of themembrane-associated phospholipase C by the VI-receptoris mediated by GTP (G-) binding proteins (Bojanic and Fain,1986; Fitzgerald et al., 1986; Fishman et al., 1987), andhence is analogous to V2-receptor activation of adenylatecyclase by Gs (Jard, 1983; Skorecki et al., 1986). Althoughthe G-protein complexes themselves have been relatively wellcharacterized (Gilman, 1987; Casey and Gilman, 1988) their

Oxford University Press

exact mechanistic role in signal transduction remains unclear.Early models to explain receptor-mediated cyclase activationpropose that lateral mobility of the hormone -receptorcomplex in the plasma membrane lipid bilayer is themechanism by which adenylate cyclase comes into contactwith, and is activated by the ligand -receptor complex(Cuatrecasas, 1974; De Meyts et al., 1976; Kahn, 1976;Tolkovsky and Levitzki, 1978). Recent work from thislaboratory (Jans et al., 1989), demonstrating a temperaturedependence of the fraction of mobile V2-receptors with thehighest mobility at 37°C, is consistent with such a model,arguing for a physiological role of receptor lateral diffusionin cyclase-mediated signal transduction. The largelyimmobile receptors for insulin and epidermal growth factor(Schlessinger et al., 1978; Zidovetzki et al., 1981; Reeset al., 1984) do not require interaction with other membraneproteins such as G-proteins to effect signal transduction andhence are different in this respect. Recent data from the ,B-adrenergic receptor- adenylate cyclase (Ransnas and Insel,1988; Ransnas et al., 1989) and other (Stryer and Bourne,1986; Lynch et al., 1986; McAndle et al., 1988) systemsindicate that agonist binding to receptor promotes theredistribution of the Gs,, into the cytosolic fraction. Thisimplies that receptor lateral diffusion-mediated interactionwith G-proteins may be the rate-limiting step in signaltransduction, all subsequent events occurring more rapidlyin the aqueous phase (Peters, 1988; Chabre, 1987).

Furthering our work with G-protein-coupled receptors, thepresent study reports photobleaching measurements of thelateral mobility of the VI-receptor in A7r5 rat smoothmuscle cells (Kimes and Brandt, 1976). This cell line is foundhere to have a similar receptor density to that of LLC-PK1renal epithelial cells (V2-receptor). The same techniques asthose used for the V2-receptor, as well as the samerhodamine-labelled analogue of vasopressin (Jans et al.,1989), could be employed to measure VI-receptor lateralmobility, enabling a comparison of the two systems. TheVI-receptor showed an apparent lateral diffusion coefficientof 2.9 to 5.1 x 10-10 cm2/s over the temperature range13 -37°C, markedly higher than comparable values for theV2-receptor which range from very low (unable to bedetermined) at 100C to 3 x 10-10 cm2/s at 370C (Janset al., 1989). Also in contrast to the V2-receptor, theVI-receptor showed negligible dependence of the fractionof mobile receptors (f) on temperature (f = 0.36-0.5 overthe same temperature range). These f values correspondedwell with the extent of VI-receptor internalization, whichis very rapid in A7r5 cells even at room temperature.

ResultsBinding properties of the Vl-receptor of A7r5 cellsThe binding affinity of the VI-receptor of A7r5 cells for[Arg8]-vasopressin was initially determined by incubatingcell monolayers with increasing concentrations of [3H]AVP

2693

D.A.Jans, R.Peters and F.Fahrenholz

in the absence and presence of a 100-fold excess of AVP(Figure 1). Scatchard analysis revealed a homogeneouspopulation of receptor sites of high affinity (KD = 1.83 ±0.02 x 10-9 M) with Bmax of 0.386 pmol/mg.The kinetics of [3H]AVP binding and internalization at

37°C, 22°C and 4°C were examined in monolayers of A7r5cells (Figure 2). Internalized ligand was determined byincubating cells, subsequent to [3H]AVP binding, with

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Fig. 1. Specific binding of [3H]AVP by A7r5 smooth muscle cells.Cell monolayers (80-90% confluent) were washed and incubated withincreasing concentrations of [3H]AVP in the absence and presence of a

100-fold excess of AVP (non-specific binding) for 30 min at 37°C.Unbound ligand was removed by washing at 4°C subsequent tobinding, and cells lysed in 0.1% Triton X-100 in NaCI/Pi. Cellextracts were both counted for radioactivity and analysed for proteinconcentration (Bradford, 1976). Results are the means (SEM,indicated) from a single typical experiment, representative of a seriesof similar experiments. The inset is a Scatchard plot of the data.

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200 mM Gly-HCl, pH 3, 200 mM NaCl for 3 min at 4°C(Segaloff and Ascoli, 1981; Schneider et al., 1988; Janset al., 1989). Both pH 3-dissociated-(dotted line, Figure 2)and cell-associated-radioactive ligand were measured.Negligible internalization occurred at 4°C even after 2 h(Figure 2). In contrast, internalization was very rapid at both37°C and 22°C, accounting for about 70% of total boundligand at steady state (Figure 2). Total specific binding wasmaximal after about 8.5 and 14 min at 37°C and 22°Crespectively, with tI/2 at about 1.8 and 4.1 min respectively.Internalization was maximal after about 12 and 30 minrespectively, with t1/2 at 37°C and 22°C at 3.8 and 9.2 minrespectively.A7r5 cells thus possess comparable receptor densities to

LLC-PKI cells (about 220 fmol/mg); however, internaliza-tion of the VI-receptor occurs notably faster than that of theV2-receptor (t1/2 = 14 min at 37°C; Lester et al., 1985;Jans et al., 1989). The kinetics and extent of [3H]AVPinternalization reported here for the VI-receptor of A7r5cells are quite comparable to those published for primaryrat hepatocytes (Fishman et al., 1985). VI-receptor re-cycling has been reported (tI12 = about 10 min, subsequentto 60 min down-regulation of receptors with AVP (Fishmanet al., 1985) but was not quantified here.

Binding and biological properties of rhodamine-labelledvasopressin in A7r5 cellsThe binding affinity of the rhodamine-labelled vasopressinanalogue 1-deamino[8-lysine(N6-tetramethylrhodamyl-aminothiocarbonyl)]-vasopressin (TR-LVP) was determinedfor the V I-receptor of A7r cells by incubating monolayerswith 10-8 M [H]AVP and various concentrations of non-radioactive TR-LVP or AVP ( Figure 3). The estimated KDvalue for TR-LVP was 3.07 i 0.82 x 10-8 M (n = 3),about 10 times that for AVP (2.83 0.05 x 10-9 M,n = 3). AVP has been reported to induce inositol1,4,5-trisphosphate (UP3) turnover in A7r5 cells (Doyle and

time (minutes)

Fig. 2. Time course and temperature dependence of [3H]AVP binding to A7r5 smooth muscle cells. Cell monolayers (80-90% confluent) were

washed and incubated with 10-8 M [3H]AVP in the absence and presence of 10-6 M AVP (non-specific binding) for various times at the indicated

temperature. Duplicate cultures were then analysed for total binding ( -*0) as described in the legend to Figure 1, or for internalized ligand

(Ol- El) as described in Materials and methods. In the latter case, cells were treated for an additional 3 min at 4°C with 200 mM Gly-HCI, pH 3,200 mM NaCl, and both cell associated (O [O1) and pH 3-dissociated (A---A) ligand determined. Results represent the mean values (SEM

indicated) of a single typical experiment, from a series of similar experiments.

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Lateral mobility of the vasopressin V1 -receptor

Ruegg, 1985) as well as Ca2+ transients (Doyle and Ruegg,1985; Ruegg et al., 1985).TR-LVP, which is biologically active in the

V2-receptor/adenylate cyclase system (Jans et al., 1989)was tested for the aforementioned activities in A7r5 cells.Concentration-dependent IP3 formation was observed inresponse to both AVP and TR-LVP (Figure 4), half-maximalresponse being induced at about 6 x 10-9 M and4 x 10-8 M respectively. This 7-fold difference in con-centration dependence was accordingly similar to that(10-fold) between the binding affinities of the two ligands.TR-LVP was also observed to be as potent as AVP ininducing rapid transient elevation of intracellular Ca2+ inA7r5 cells, using fura-2 as a fluorescent Ca2' probe (datanot shown). As expected, neither AVP nor TR-LVP(l0-7 M) induced cAMP production in A7r5 cells (notshown), in contrast to their marked effects in LLC-PKIcells, consistent with the lack of V2-receptors in A7r5 cells.

The rhodamine-labelled vasopressin analogue as afluorescent label specific for the V1-receptor of A 7r5cellsThe fluorescence microphotolysis (photobleaching) apparatus(Peters, 1986) was employed to assess specificity of TR-LVP binding to the VI-receptor of living A7r5 cells. Cellmonolayers were incubated with 10-7 M TR-LVP(maximal binding-not shown) in the absence or presence(non-specific binding) of 10-5 M AVP, washed, mountedin ligand-free medium, and cell-associated fluorescencemeasured in circular areas of plasma membrane of 2 itmradius. Maintaining the measurement parameters constant,parallel series of measurements were performed to derivevalues for non-specifically bound- (above) and auto- (cellsincubated in the absence of ligand) fluorescence. Table Ishows results from a typical experiment, demonstrating thatsubtraction of non-specific fluorescence from totalfluorescence enabled the quantification of specifically boundfluorescence due to TR-LVP binding to the VI-receptor of

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Fig. 3. Competition of [3H]AVP binding to A7r5 cells by AVP andthe rhodamine-labelled vasopressin analogue TR-LVP. Monolayerswere incubated with 10-8 M [3H]AVP and the indicatedconcentrations of AVP (El -OI) or TR-LVP (0 -0) for 30 min at37°C. Cells were washed, lysed and counted as described in thelegend to Figure 1. Results represent the means (SEM as indicated)for a single typical experiment performed in triplicate from a series ofsimilar experiments.

A7r5 cells. Cells preincubated with ligand at 4°C, as opposedto at 220C, exhibited higher specifically bound fluorescencemeasured at 22°C (Table I). At low frequency (estimatedat about 4 and 7% respectively of the sites in cellspreincubated with ligand at 4°C and 22°C, as determinedby random single-cell measurements) highly fluorescent siteswere detected (Table I). Examined in more detail, these siteswere found to be of higher fluorescence in the case of cellspreincubated with TR-LVP at 22°C than those preincubatedat 4°C. This implied the existence of highly fluorescentreceptor aggregates, which appeared to increase both inprevalence and fluorescence intensity with time at 22°C,interestingly paralleling the kinetics of internalization (seeFigure 2). Since all measurements were performed at 22°C,the cells preincubated with TR-LVP at 22°C, as comparedto those preincubated at 4°C, can effectively be consideredas having been incubated for 10-15 min longer at 22°C;hence, the higher receptor aggregation and internalizationapparent.These conclusions were supported by video-enhanced

fluorescence microscopy of cells washed subsequent topreincubation with TR-LVP at 22°C (Figure 5). A cleardifference in the intensity of cell-associated fluorescence wasevident between cells incubated with 10-7 M TR-LVP inabsence (Figure SB) and presence (non-specific binding) of10-5 M AVP (Figure SD). Fluorescence was largelyassociated with aggregates in the cells incubated with ligandat 22°C (Figure SB). Cells preincubated with TR-LVP at4°C showed a more diffuse pattern of fluorescence,compared to cells preincubated at 22°C, with fewer highlyfluorescent aggregates (not shown), consistent with the aboveresults.

Lateral mobility of the V1-receptorThe photobleaching technique (Peters, et al., 1974; Peters,1986) was used to measure the lateral mobility of theVI-receptor in membranes of living cells of the A7rS cellline (see Jans et al., 1989, for details of the comparable studyfor the V2-receptor of LLC-PK1 cells). As in the previousstudy, fluorescence intensity was monitored at short intervals

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Fig. 4. Biological activity of TR-LVP. Concentration dependence of

inositol 1,4,5-trisphosphate (1P3) formation of A7r5 cells in response to

AVP (--0) or TR-LVP ([1 -[1). Cell monolayers were incubated

for 30 s at 37°C with the indicated ligand concentrations and then

washed and cell-free extracts prepared and assayed for IP3 accordingto Palmer et al. (1986) (see Materials and methods). Results are

duplicate deteminations from a single typical experiment.

2695

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D.A.Jans, R.Peters and F.Fahrenholz

Table I. Binding of the rhodamine-labelled vasopressin analogue TR-LVP to A7r5 cells as determined by single-cell fluorescence measurements

Preincubation Fluorescence (arbitrary units) measured at 22oCaPreincubation conditions10 min 22°C 60 min 4°C

(a) No ligand 5525 ± 510 (17) 5742 i 530 (19)(b) 10-7 M TR-LVP 10797 + 1523 (28) 12537 ± 1375 (24)(C) i0-7 M TR-LVP + i0-5 M AVP 7726 f 667 (21) 5841 + 351 (21)(d) (b) -(c) Specific fluorescence 3071 6696

Highly fluorescent sitesb(e) 10-7 M TR-LVP 54481 11170 (10) 35838 ± 5880 (9)(f) 10- M TR-LVP + i0-5 M AVP 15082 + 2455 (8) 16577 f 3006 (8)(g) (e)-(f) Specific fluorescence 39399 19261

aCells were grown for 3 days on coverslips in 12-well Costar-plates and then treated for the time and temperature indicated in serum-free medium.Cells were then washed four times with NaCl/Pi, prior to mounting in serum-free medium at room temperature. Measurements of fluorescenceintensity were performed at room temperature as described in the text. Values represent the mean + the standard error of the mean, with n thenumber of measurements, in parenthesis.bSee text for details. The ratio of highly fluorescent sites to 'normally' fluorescent sites was about 0.07 and 0.04 for cells preincubated with TR-LVPat 22°C and 4'C respectively as estimated by random single cell measurements (n > 60).

of the fluorescence recovery process, rather than in the usualcontinuous fashion, to avoid the occurrence of (unintentional)bleaching during the course of the measurements.Maintaining the measurement parameters constant, parallelseries of measurements were performed on several differentcells for each experimental treatment, with measurementsfor each time interval pooled and averaged.

Figure 6 shows a typical series of measurements ofVI-receptor lateral diffusion at 22°C subsequent toprelabelling with 10-7 M TR-LVP for 10 min at 22°C(Figure 6A). Measurements of the recovery of total boundfluorescence after photobleaching were performed (Figure6A, triangles) and, under identical measurement conditions,of the recovery of non-specifically bound fluorescence afterphotobleaching (Figure 6A, squares), where A7r5 cellspreincubated with 10-7 M TR-LVP together with 10-5 MAVP were used. The values of the photobleaching recoveryfor non-specific fluorescence were subtracted from those fortotal bound fluorescence to yield the photobleaching recoveryof specifically bound fluorescence (Figure 6B, circles). Thelatter were then evaluated for two components; a mobilefraction f, with an apparent lateral diffusion coefficient, D,and an 'immobile' fraction (D < 10-12 cm2/s) (Axelrodet al., 1976). Experimental data were fitted by theoreticalcurves (Figure 6B) according to Soumpasis (1983) and a non-linear least square algorithm.

Results of lateral mobility measurements of theVI-receptor of A7r5 cells are summarized in Table II. Amarked temperature dependence was evident in the apparentlateral diffusion coefficient of the VI-receptor, whereby Dwas lowest (2.85 x 10-10 cm2/s) at the lowest measuringtemperature (13°C), and highest at 37°C (5.13 x 10-lcm2/s). Both the nature of the temperature dependence, andvalue of D here are quite comparable to published resultsfor integral membrane proteins and in particular membranereceptors (see Edidin, 1987; Hillman and Schlessinger,1982). The D values, however, are notably higher than thosedetermined for the V2-receptor (Jans et al., 1989). Also, incontrast to the V2-receptor, no marked temperaturedependence of the receptor mobile fraction was evident, franging from 0.36 to 0.52 over the temperature range13-37°C (Table II). The f values are both 22°C and 37°C

Fig. 5. Visualization of TR-LVP binding to A7rS cells. Cells wereincubated for 10 min with ligand at 22°C, washed twice, and mountedin serum-free medium. Cells were photographed using a 1OOx oilimmersion objective. (A and B) A7rS cells preincubated with 10-7 MTR-LVP and photographed under normal (A) or fluorescent (B)illumination. (C and D) A7rS cells preincubated with 10-7 MTR-LVP and 10-5 M AVP under normal (C) or fluorescent (D)illumination.

here are also significantly lower than those reported for theV2-receptor, and thereby reflect the extent of internalizationof the VI-receptor (Figure 2). Receptor internalizationpresumably results in rapid receptor immobilization, and arelatively low receptor mobile fraction. The f values hereare of course comparable to those reported for other rapidlyinternalized receptors such as those for insulin and epidermalgrowth factor (Schlessinger et al., 1978; Zidovetzki et al.,1981). Although one might have expected a differencebetween f values measured at 22°C on cells preincubatedwith ligand at either 4°C or 22°C as the result of rapidVI-receptor internalization (see Figure 2 and Table I andaccompanying Discussion in the text), this was not the case(Table II). This was probably due to the long duration ofthe diffusion measurements (30-40 min for a series of 5-6individual measurements), which would mask any

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Lateral mobility of the vasopressin Vl-receptor

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Fig. 6. Lateral mobility of the VI-receptor in the plasma membranesof A7r5 cells at 22°C. Cells were washed subsequent to a 10 minincubation with 10-7 M TR-LVP at 220C, and mounted in serum-freemedium. Measurements were performed as described in the text for a

further 5-24 min at 22°C. (A) A-A, total bound fluorescence (A7r5cells treated with 10-7 M TR-LVP); and *-*, non-specificallybound fluorescence (A7r5 cells treated with 10-7 M TR-LVP togetherwith l0-5 M AVP). Each point represents the average of at least 6separate measurements, with the standard error of the mean indicated.(B) specific fluorescence (0 - 0), representing non-specific subtractedfrom total fluorescence. The full line repesents the least-square best fit(see text for details).

differences resulting from 10-15 min longer incubation (andinternalization) at 22°C in the case of cells preincubated withTR-LVP at 22°C, compared to those preincubated at 4°C.A few measurements (not shown) were made ofVI-receptor lateral mobility in the 'highly fluorescent sites'(above). The values of f for cells preincubated with TR-LVPat 4°C and 22°C and then measured at 22°C were 0.3and 0.2 respectively (D values not able to be determined),consistent with the idea that these sites are largely im-mobile receptor aggregates, probably related to receptorinternalization.

DiscussionThis study reports the first measurement of the lateralmobility of a polypeptide hormone receptor which is coupledto G-protein-mediated activation of phosphoinositol metabolism.The results here for the VI-receptor of A7r5 cells comple-ment those from our previous study for the V2-receptor ofLLC-PKI cells (Jans et al., 1989) and enable a comparisonof the two G-protein-coupled vasopressin receptors in thecontext of their analogous but different signal transductionmechanisms. This comparison of Vl- and V2-receptors is

made possible through the fact that both A7r5 and LLC-PK, cells have similar vasopressin receptor densities, andthat the TR-LVP fluorescent ligand has reasonably highreceptor affinity and specificity, as well as biological activity,in both systems. In similar fashion to our previousV2-receptor study, we were able to use non-specificfluorescence controls to enable a quantitative and dependableestimate of specific VI-receptor lateral mobility in A7r5plasma membranes, and overcome the problems of weakfluorescence intensity (only one chromophore per ligandmolecule) and low receptor densities (about 2000 receptorsper illuminated area). This study shows a clear dependenceof the apparent lateral diffusion coefficient of theVI-receptor on temperature, being lowest (2.85 x 10-1Ocm2/s) at the lowest measuring temperature (13°C) andhighest at 37°C (5.13 x 10-1 cm2/s). These values arecomparable to those reported for other polypeptide hormonereceptors such as those for insulin and EGF (Hillman andSchlessinger, 1982; Schlessinger et al., 1978) and aresignificantly higher than the comparable values for theV2-receptor (e.g. 3.0 x 10-10 cm /s at 37°C). That D ishighest at 37°C argues for its physiologicial importance anda possible role in signal transduction. This idea is supportedby the more rapid kinetics of signal transduction in theVI-receptor (and insulin and EGF receptor) systems com-pared to the V2-receptor-adenylate cyclase system(Steinberg et al., 1979; Jans et al., 1987b). The latter hasa lower D value, and markedly slower activation kineticswhereby intracellular cAMP is half-maximal about 120 safter AVP addition at 37°C in LLC-PK1 cells (manuscriptin preparation) whereas IP3 formation peaks 30 s after AVPaddition in A7r5 cells and intracellular Ca2+ accumulationis half-maximal in less than 60 s (H.J.Knot, personalcommunication). It accordingly seems reasonable tohypothesize that the rate of diffusion of membrane-associatedreceptors may play a determining role in signal transduc-tion kinetics, especially in systems where interaction withother membrane-associated proteins (G-proteins) is required.The EGF system requires no interaction with G-proteins;but probably requires receptor oligomerization (receptor-receptor collision) to effect signal transduction through inter-molecular autophosphorylation (Schlessinger, 1988). Theinsulin receptor also appears to display receptor aggrega-tion (Johnson et al., 1988). A high D value in these systemswould bring about rapid receptor aggregation (Schlessingeret al., 1978; Hillman and Schlessinger, 1982) and con-comitant rapid signal transduction, consistent with the ideaof receptor lateral mobility being of physiological import-ance in the respective signal transduction processes.We have argued that the receptor mobile fraction in the

V2-receptor system, being highest at 37°C and lowest at10°C, is of physiological importance in terms of themechanism of signal transduction in the adenylate cyclasesystem. The G-protein coupled VI-receptor here exhibits nomarked dependence of f on temperature. The f values werebetween 0.36 and 0.52 over the range 37°C-13°C, muchlower than those for the V2-receptor (f = 0.91 at 37°C).The fact that low f values were determined for theVI-receptor at 37°C or 22°C is most likely to be explicablein terms of the rapid internalization kinetics-after only15 mmn at 22°C, more than 50r% of AVP bound isinternalized. It is understandable, under these conditions,

2697

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D.A.Jans, R.Peters and F.Fahrenholz

Table II. Lateral mobility of the V1-receptor in membranes of A7r5 cells

Temperature of: Parameter of mobilitya

Preincubation Measurement D fwith ligandb (10- I0cm2/s)

370C 370C 5.13 + 0.66 0.36 + 0.02 (4)220C 220C 3.58 + 0.46 0.50 + 0.01 (5)40C 220C 3.51 + 0.50 0.53 + 0.01 (5)40C 130C 2.85 + 0.42 0.44 a 0.03 (3)

aValues are the mean SEM (n, the number of experiments in parenthesis) for D, apparent lateral diffusion coefficient and f the mobile fraction.Each experiment represented at least 5 single-cell measurements (over about 30 min) for each of total bound- and non-specific-fluorescence,respectively (see Figure 6).bPreincubations with ligand were for 10 min at 370C or 220C, and 60 min at 40C, prior to washing with NaCI/Pi at the measuring temperature.

with the time-consuming nature of the diffusionmeasurements, that no high f values could be detected.Similar results for the EGF receptor (Zidovetzki et al., 1981;Rees et al., 1984; Schlessinger et al., 1978) can berationalized in these terms. VI-receptor aggregation wasalso able to be visualized here even at 22°C (Figure 5, andnot shown) consistent with the idea of receptor aggregation,immobilization, and internalization occurring at a much fasterrate than in the V2-receptor system. For the latter system,where the slower receptor internalization kinetics permit suchan analysis, it is possible to demonstrate that internalizationparallels V2-receptor immobilization and reduction of f.Measurements of V2-receptor f in LLC-PK1 renal epithelialcells at various times after ligand addition revealed a directcorrelation between the immobile receptor fraction andinternalized receptor (unpublished observation). Thatreceptor mobile fraction and internalization are inverselyrelated has also been demonstrated for the EGF and insulinreceptors using NaN3, an inhibitor of receptorinternalization (Schlessinger et al., 1978). Treated cellsexhibit significantly higher f values for both receptors in thepresence of NaN3, compared to in its absence (Schlessingeret al., 1978).Hillman and Schlessinger (1982) (see also Schlessinger,

1989) have proposed that EGF receptor lateral diffusion ismechanistically important in receptor endocytosis, in thatthe EGF receptor must migrate to coated pit regions of thecell plasma membrane in order to undergo internalization.This introduces the interesting possibility that receptor lateralmobility not only plays a role in signal transduction itself,by bringing proteins into contact with one another in theplasma membrane lipid bilayer; but that lateral diffusion ofreceptors may also be important in receptor down-regulationand endocytosis. Direct investigation of the role of receptorlateral mobility in both signal transduction and receptordesensitization is currently the focus of future work in thislaboratory.

Materials and methodsMaterials were as described previously (Jans et al., 1986, 1987a,b, 1989).The rhodamine-labelled analogue of vasopressin, 1-deamino[8-lysine(N6tetramethylrhodamylaminothiocarbonyl)]vaspressin (TR-LVP) wasprepared as described (Jans et al., 1989). Cells of the A7r5 rat smooth musclecell line (Kimes and Brandt, 1976) were cultured in DMEM supplementedwith 10% (v/v) fetal calf serum 0.2 mg/ml streptomycin and 50 U/mlpenicillin.

Vasopressin bindingVasopressin binding was measured on whole cell monolayers as describedfor LLC-PKI cells (Jans et al., 1989). Internalized hormone was

2698

determined using a 3 min treatment with 200 mM Gly-HCI, pH 3, 200 mMNaCI subsequent to binding incubation as previously described (Segaloffand Ascoli, 1981; Schneider et al., 1988; Jans et al., 1989). Dissociationconstants (KD), the concentration of hormone corresponding to 50%maximal binding, were determined as described previously (Fahrenholzet al., 1985; Jans et al., 1986). Protein was estimated using the dye bindingassay of Bradford (1976) with BSA as standard.

Fluorescence measurementsCells to be used for fluorescence and lateral diffusion measurements weregrown on coverslips (15 x 15 mm) for 3-4 days to about 50% confluence.After incubation with ligand, cells were washed with NaCI/Pi (containing0.5 mg/ml BSA), and mounted in the incubation medium in the absenceof ligand. The methods used in measurements of fluorescence intensity andthe fluorescence microphotolysis apparatus used have been describedpreviously in detail (Peters, 1986).

Inositol 1,4,5-trisphosphate OP3) determinationsCells were grown to 90-95% confluence in 24-well Costar-plates, treatedfor 30 s with ligand and the incubation terminated by addition of 200 Id20% perchloric acid (4°C). After 20 min on ice, supernatants were titratedto pH 7.5 with KOH, and then centrifuged for 20 min at 4000 r.p.m. Super-natants were then analysed for IP3 using the competitive binding assay kitfrom Amersham (Palmer et al., 1986).

AcknowledgementsThe authors are grateful to Dr H.J.Knot, Sandoz, Basel, Switzerland forthe IP3 and Ca2+ determinations; and to Patricia Jans for ever-cheerful andcapable technical assistance in the face of eternally petulant collaborators.This work was supported by grants of the Deutsche Forschungsgemeinschaftto F.F. and R.P., respectively.

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Received on March 19, 1990; revised on May 18, 1990

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