The carboxyl terminus of the hamster β-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

Cell, Vol. 49, 655-863,

June

19, 1987. Copyright

0 1987 by Cell Press

The Carboxyl Terminus of the Hamster p-Adrenergic Receptor Expressed in Mouse L Cells Is Not Required for Receptor Sequestration Catherine D. Strader: Irving S. Sigal:t Allan D. Blake: Anne H. Cheung,’ R. Bruce Register,t Elaine Rands,t Barbara A. Zemcik: Mari Rios Candelore: and Richard A. F. Dixont + Department of Biochemistry and Molecular Biology Merck Sharp and Dohme Research Laboratories Rahway, New Jersey 07065 t Department of Virus and Cell Biology Research Merck Sharp and Dohme Research Laboratories West Point, Pennsylvania 19486

Summary The structural basis for agonist-mediated sequestration and desensitization of the P-adrenergic receptor (PAR) was examined by oligonucleotide-directed mutagenesis of the hamster PAR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of PAR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, PAR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for CAMPdependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the PAR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to G, and sequestration. Introduction The actions of a variety of hormones and neurotransmitters are mediated through interaction with specific receptors that couple to guanine nucleotide-binding proteins, G-proteins. The best characterized of these receptors is the f3-adrenergic receptor @AR), which, upon binding catecholamines, interacts with the G-protein G, to stimulate adenylate cyclase activity. The genes encoding both the mammalian (Dixon et al., 1986; Kobilka et al., 1987) and the avian (Yarden et al., 1986) 8AR and the porcine brain (Kubo et al., 1986a) and cardiac (Kubo et al., 1986b) muscarinic cholinergic receptors have been cloned, revealing amino acid sequence homology among these receptor proteins and with the visual opsins (Nathans and Hogness, 1983) which act by stimulating the G-protein transducin. Upon prolonged exposure to agonists, many of these G-protein-linked receptors exhibit a reduction in responsiveness to subsequent agonist challenge referred to as desensitization. The desensitization of these receptors has important physiological and pharmacological consequences. Desensitization of the 8AR involves a loss of

agonist-responsive adenylate cyclase activity and is of two basic types (for review see Perkins, 1983; Sibley and Lefkowitz, 1985). Heterologous desensitization results in a loss of activity of the PAR, as well as of other receptors acting through G,, to stimulate adenylate cyclase. Homologous desensitization, on the other hand, is specific for the BAR. Despite extensive study, the mechanisms for both heterologous and homologous desensitization remain illdefined. In most systems, desensitization is accompanied by receptor sequestration, the reduction in the number of receptors at the cell surface. Some experimental evidence indicates that the receptors lost from the surface are sequestered to a specific membrane fraction and are later degraded (Su et al., 1980; Waldo et al., 1983; Kassis and Sullivan, 1986). This latter process is referred to as downregulation. Both homologous and heterologous desensitization have been correlated with PAR phosphorylation in some cell types, and this phosphorylation has been postulated to be the causative event in these systems (Sibley et al., 1984,1986). It has been hypothesized that the phosphorylation involved in heterologous desensitization is mediated by CAMP-dependent protein kinase (PKA), while that involved in homologous desensitization is mediated by a receptor-specific kinase, designated the 8AR kinase, or BARK (Benovic et al., 1986a). Two putative PKA sites, amino acid residues 259-262 and 343-348 have been noted in the sequence of the mammalian PAR (Dixon et al., 1986). These regions share the sequences of known substrates for PKA, which have the consensus sequence Lys/Arg-Arg-X-(X)-Ser-X-, where a serine in proximity to several basic amino acids represents a good target for the enzyme (Feramisco et al., 1980). Synthetic peptides corresponding to these sequences act as substrates for the kinase in vitro (Blake and Strader, unpublished results). The substrate specificity for PARK has not yet been established. In light of the homology between the BAR and rhodopsin (Dixon et al., 1986) the C-terminal region of the BAR has been suggested as a possible substrate for PARK (Benovic et al., 1986b) by analogy with the interaction between light-activated rhodopsin and rhodopsin kinase (Hargrave et al.,1980). In that system, phosphorylation of serines near the C-terminus of rhodopsin stimulates its binding to the S-antigen arrestin, reducing the ability of rhodopsin to interact with transducin to stimulate phosphodiesterase (Wilden et al., 1986). It is possible that a similar mechanism may be acting in the 8-adrenergic system. In the present study, we have examined the importance of specific regions of the hamster 8AR in desensitization. The gene encoding the hamster B2AR was expressed in a mammalian cell system in which agonist-mediated sequestration and desensitization were studied. Mutations that resulted in the deletion of the C-terminus of the PAR and of the putative PKA substrate sites, as well as a mutation that interfered with the coupling of the receptor to G,, were introduced into the receptor. The ability of the recep-

Cdl 856

0

BAR

D (239.272) BAR D(k ,) PAR D(k 2) PAR Dhz 1 PAR r(395) BAR T(354) PAR D(klp)TW)PAR

Figure

10,o

I

0

I

NH2

COOH

I I I 1 I , I

1. Mutagenesis

of the 6AR

The solid line at the top represents the sequence of the wild-type PAR, with the solid boxes indicating the relative positions of the hydrophobic regions of the receptor. Written above the drawing, i represents regions of the receptor that would be postulated to be exposed inside the cell, and o, regions exposed outside the cell, based on a model in which the N-terminus of the 8AR is exposed extracellularly and each of the hydrophobic regions traverses the membrane once. Seven deletion mutations of the 8AR used in this study are diagrammed below the wild-type BAR, with the positions of the deletions indicated by gaps in the linear sequence. The actual residues removed in the mutations are as follows: D(239-272) PAR, 239-272; D(k,) BAR, 259-262; D(kd BAR, 343-348; D(k,s) BAR, 259-262 and 343-348; T(395) BAR, 395-418; T(354) BAR, 354-418; D(kls)T(395) BAR, 259-262, 343-346, and 395-418. Mutants D(229-236) BAR, D(250-259) BAR, D(239-272) 8AR, D(k,) PAR, D(ks) BAR, and T(354) 6AR have been previously described (Dixon et al., 1987) and mutants D(k,d BAR, D(226-239) PAR, T(395) BAR, and D(ktdT(395) BAR were prepared for this study as described in Experimental Procedures.

tor to interact with G, was found to correlate with its ability to undergo agonist-mediated sequestration. In contrast, this sequestration process did not require the presence of the C-terminal region or the putative PKA sites of the PAR. Results Mutagenesis and Expression The structural requirements for desensitization were explored using a series of mutant PAR having the deletions listed in Figure 1 and Table 1. In mutant D(239-272) PAR, a large peptide segment that would be predicted to correspond to an intracellular region of the receptor was deleted. This region of the protein was further examined

Table

1. Properties

of PAR Mutants lsoproterenol

Adenylate

Kd (PM)

GO W4

kc1 OW

FoldStim.

ND 70 100 ND 10 100 ND ND ND 75 150 ND

ND 30 50 30 10 20 20 30 20 30 30 30

ND 20 5 IO ND 30 30 30 ND 20 10 30

0 3 3 7 0 3 4 4 3 3 2 2

‘2%CYP Protein

Bmax

Control BAR’ D(229-236) D(238-251) D(239-272) D(250-259) D(h) BAR” D(b) PAR’ Wd BAR T(395) 8AR T(354) BAR’ DWWW

0 110 840 900 200 180 200 400 230 130 350 230

PAR’ PAR PAR’ 8AR’

PAR

with smaller deletions, D(229-236) PAR, D(236-251) PAR, and D(250-259) BAR. In a second group of mutants the sequences corresponding to the two consensus sites for PKA substrates, residues 259-262 and 343-346, were deleted individually, forming D(k,) PAR and D(kd PAR, respectively. In mutant D(k,s) PAR, both of these sites were deleted together. In addition, two mutants were generated having C-terminal truncations, T(395) BAR and T(354) PAR. Finally, mutant D(k12)T(395) PAR combined the deletions of both putative PKA sites with the deletion of the C-terminus. The functional properties of these mutant receptors, when expressed in mouse L cells, are listed in Table 1. As we have previously reported (Dixon et al., 1967), control L cells transfected with the expression plasmid alone did

(fmollmg)

Cyclase

Saturation binding of 1*51-CYP to membranes from clonal L cell line expressing the various mutants was measured using lo-400 pM ‘251-CYP in an assay volume of 250 ul of TME buffer containing 15-25 ug of membrane protein. Binding was performed for 90 min at 23% and bound radioactivity was determined by filtration on GFlC glass fiber filters. Nonspecific binding, defined in the presence of 10 uM alprenolol, was <5%. Data were analyzed by nonlinear regression. lsoproterenol binding was measured in competition with 35 pM r*sl-CYP in 250 ul of TME buffer, in the presence of increasing concentrations of (-)isoproterenol at a final PAR concentration of 5-7 pM for 90 min at 23%. ICSO is defined as the concentration of isoproterenol at which 50% of the ‘*r+CYP binding to the 8AR was inhibited. The relationship of the I& to the true & for in the presence of inisoproterenol has a dependence on the & of each mutant PAR for 1251-CYP. Adenylate cyclase stimulation was measured creasing concentrations of isoproterenol as described in Experimental Procedures, and the activation curves were analyzed by nonlinear regression. The maximal fold stimulation by 10-s M isoproterenol over the basal level is given in the last column of the table. Some of these data, designated with an asterisk, have been reported in another study (Dixon et al., 1987). For comparison, in that study, D(k,) f3AR is referred to as HAM(del 259-262) D(ks) PAR as HAM(del 343-348) D(239-272) 8AR as HAM(239-272). D(229-236) 6AR as HAM(229-236) D(250-259) BAR as HAM(250-259) and T(354) f3AR as HAM(trunc 354).

6-Adrenergic 857

$ 5

80

Receptor

Desensitization

stimulated adenylate cyclase activity resulted from an abnormal coupling of this receptor to G,, as evidenced by the absence of any effect of GTP on agonist binding @trader, Sigal, and Dixon, unpublished results).

1

Figure 2. lmmunoprecipitation Labeled DAR

of Mutant

and

Wild-Type

“sl-CYP-

1251-CYPSlabeled f3AR was prepared as described in Experimental Procedures. For immunoprecipitation, 25 ~1 of t*sl-CYP-labeled DAR was incubated with 10 ul of antiserum and 40 t~l of Tris-NaCI buffer for 2 hr at 23%. The antibody was then precipitated by incubation with 75 pi of saturated ammonium sulfate for 30 min at 4%, the pellet was isolated by centrifugation at 12,000 x g for 3 min, and the radioactivity incorporated into the pellet was determined with a gamma counter. Nonspecific immunoprecipitation, accounting for approximately 10% of the total, was determined with nonimmune serum and was subtracted from the values shown in the Figure. The hatched bars represent the immunoprecipitation of tz51-CYP-labeled f3AR with a polyclonal antibody to a synthetic peptide representing amino acids 224-242 of the hamster f3AR, while the stipled bars represent immunoprecipitation by a polyclonal antibody to a synthetic peptide representing the C-terminus of the f3AR (amino acids 404-418). Peptide synthesis and antibody production are described in Experimental Procedures. The PAR mutant used for each immunoprecipitation is printed along the bottom of the Figure. The total ‘251-CYP-labeled BAR added to each incubation was as follows: wild-type BAR, 14,000 cpm; D(239-272) PAR, 13,500 cpm; D(k,a) PAR, 8,ooO cpm; D(k,,)T(395) DAR, 13,500 cpm; T(395) @AR, 7,000 cpm; T(354) PAR, 7,000 cpm.

not express detectable levels of 6-adrenergic ligand binding or of isoproterenol-stimulated adenylate cyclase activity. Introduction of DNA encoding the wild-type or the mutant PAR into the expression system led to the isolation of clonal cell lines expressing functional PAR, as assessed by the binding of the 6-adrenergic antagonist lz51-cyanopindolol (1251-CYP) or of the agonist isoproterenol. As seen in Table 1, all of the mutant receptors bound both the agonist and the antagonist with affinities similar to those of the wild-type protein. All of the mutants except for D(239-272) f3AR stimulated adenylate cyclase with a K,,, comparable to that of the wild-type protein. As discussed previously (Dixon et al., 1987), the maximal level of stimulation varied among different clonal cell lines expressing the same mutant and did not relate to the specific mutant involved. In all cases where isoproterenol-stimulated cyclase activity was observed, the maximal stimulation by isoproterenol corresponded to the maximal level of stimulation seen by NaF (data not shown). As described previously, the mutant D(239-272) BAR did not stimulate adenylate cyclase even at high concentrations of isoproterenol, even though this mutant binds isoproterenol with high affinity (Dixon et al., 1987). This lack of agonist-

Immunological Characterization of Mutant Proteins The authenticity of the expressed mutant receptors was confirmed by immunoprecipitation using site-specific antisera. The PAR in the L cell membranes was labeled with 12%CYR solubilized, and immunoprecipitated with antibodies recognizing two different regions of the hamster DAR. One antibody was raised to a synthetic peptide corresponding to residues 224-242 of the DAR. As shown in Figure 2, this antibody immunoprecipitated the wild-type PAR and several of the mutant proteins, but did not immunoprecipitate D(239-272) PAR, confirming that a portion of the PAR overlapping with residues 224-242 was deleted in this mutant. An antibody to a synthetic peptide corresponding to the C-terminus of the f3AR was able to immunoprecipitate the wild-type receptor and D(239-272) PAR, as well as D(k12) PAR. However, this antibody did not recognize T(395) PAR, T(354) PAR, or D(k12)T(395) DAR, confirming the deletion of the C-terminus in these mutants. The expression and normal glycosylation of some of these mutants has previously been shown by protein immunoblotting (Dixon et al., 1987). Isoproterenol-Mediated Sequestration of Receptors The sequestration of the mutant receptors from the cell surface upon exposure to isoproterenol was measured using the hydrophilic antagonist, 13H]CGP 12177, which binds only at the surface of intact cells (Hertel et al., 1983). As shown in Figure 3A, treatment of cells expressing the wild-type PAR with isoproterenol resulted in a rapid loss of receptors from the cell surface, with a final loss of 600/o80% of the total surface receptors within 30 min. Deletion of the C-terminus from the PAR did not dramatically affect receptor sequestration, as seen in Figure 3A. The additional deletion of the putative PKA sites also did not affect the agonist-mediated sequestration process (Figure 3A). That this loss of 13H]CGP binding sites corresponded to a loss of cell surface receptors and not to the irreversible binding of isoproterenol to the 6AR was shown by performing the isoproterenol incubation at 4% a temperature permissive for ligand binding to the receptor but not for receptor internalization. In this experiment, there was no loss of [3H]CGP binding sites during the first hour of incubation with the ligand (Figure 3A). In contrast to the rapid sequestration seen with the mutants discussed above, the number of surface receptors for the mutant D(239-272) PAR was only slightly decreased upon exposure of the cells to isoproterenol. As shown in Figure 3A, treatment of the cells expressing this mutant with isoproterenol under conditions that produced maximal sequestration of the wild-type PAR resulted in only a very slow loss of D(239-272) f3AR from the cell surface. The final loss of 40% of the total surtace receptors was achieved only after a 3 hr incubation with isoproterenol and did not increase even after a 16 hr exposure to the agonist (data not shown). Thus, the deletion of residues

Cell

058

0

15

30

me

Figure

3. Sequestration

wtth

45

~soproferevJ

of Cell-Surface

60

‘80

fmtni

incubation

time (min)

PAR

Ceils expressing the wild-type or mutant PAR, growing in monolayer culture in 24-well plates, were exposed to (-)isoproterenol or dibutyryl CAMP by addition of the compound directly to the culture media, and the cells were incubated at 37% under normal growth conditions, as described in Experimental Procedures, for the times indicated along the x-axes. Sodium metabisulfite (0.2 mM) was included with the isoproterenol to enhance its stability. At the end of the incubation, the plates were transferred to an ice bath and the monolayers were washed 3 times with ice-cold PBS. The binding of [3H]CGP 12177 (Amersham) was measured directly on the monolayer with 20 nM 13H]CGP in a total volume of 0.25 ml of PBS containing 0.1% BSA. Equilibrium binding was performed for 4 hr at 4°C after which the monolayers were washed 3 times with PBS and removed from the plate in 1% SDS. Radioactivity was counted in a liquid scintillation counter. Nonspecific binding, assessed in the presence of 10-s M alprenolol, was 5%-15% of the total bound ligand. Data shown are the means of 2-5 experiments. (A) [3H]CGP binding to cells after treatment with agonist. The cells used were expressing the following receptors, treated at 37% with isoproterenol unless otherwise noted: f3AR (filled circles), D(239-272) BAR (filled squares), T(395) f3AR (filled triangles), T(354) PAR (open triangles), and (k,a)T(395) BAR (open circles). Open squares show the effect on wild-type surface f3AR when the isoproterenol incubation was carried out at 4OC instead of 37°C. Crosses designate cells expressing wild-type PAR incubated with 10-s M PGE at JPC. (B) Comparison of the effects of 10m5 M isoproterenol (filled circles, filled triangles) and 1O-3 M dibutyryl CAMP (open circles, open triangles) on the subsequent binding of 13H]CGP to cells expressing BAR (filled circles, open circles) and D(239-272) BAR (filled triangles, open triangles).

239-272 from the BAR, which resulted in a loss of the ability of the receptor to interact productively with G,, also caused a severe attenuation of the sequestration response to agonists. The results of a 30 min exposure to isoproterenol for all of the mutants are summarized in Table 2. As observed for the wild-type receptor, other mutants either incorporating smaller deletions within the hydrophilic region 221-273 PAR, the putative PKA sites, or the C-terminus responded to isoproterenol with a rapid 70%-800/o sequestration of surface receptors.

cAMPMediated Sequestration of Receptors Treatment of cells expressing wild-type receptors with prostaglandin El (PGE) did not result in a measurable loss of surface 8AR sites (Figure 3A). The presence of PGE receptors in these cells was demonstrated by the observation of a 3-4 fold stimulation of adenylate cyclase activity by lO-5 M PGE. A small amount of PAR sequestration was seen when the cells were treated with dibutyryl CAMP, as shown in Figure 38. When 8-bromo CAMP was substituted for dibutyryl CAMP in the same experiment, no effect on sequestration was observed. The low amount of sequestration of the wild-type f3AR with dibutyryl CAMP was also seen in cells expressing D(239-272) 8AR and approxi-

mated the level of receptor loss seen with isoproterenol in these cells (Figure 38). The effects of isoproterenol and dibutyryl CAMP were not additive (data not shown). The dibutyryl CAMP-induced sequestration of the mutants having deletions of the putative PKA sites and/or the C-terminus is summarized in Table 2. In all cases the dibutyryl CAMP-induced sequestration of the mutant receptors was indistinguishable from that of the wild-type PAR. Immunofluorescent Localization The isoproterenol-induced sequestration of the PAR was also detected by immunofluorescence using a monoclonal antibody to a peptide corresponding to residues 226-239 of the hamster PAR. To observe reactivity with the antibody, which is believed to interact with an intracellular epitope, the cells were fixed and permeabilized prior to antibody treatment. As shown in Figure 4A, cells expressing the wild-type 8AR had a low level of background fluorescence in the presence of the nonimmune antibody, which was markedly enhanced with the addition of the specific monoclonal antibody. Treatment of the cells with isoproterenol prior to fixation resulted in a loss of this specific immunofluorescence, with the levels returning almost to background after a 1 hr exposure to the agonist. Cells expressing D(239-272) 8AR also showed specific immu-

&&drenergic

Receptor

Table 2. Effect

Desensitization

of lsoproterenol

or Dibutyryl

[3H]CGP Protein

BAR D(229-236) D(238-251) D(239-272) D(250-259)

BAR PAR PAR BAR

W) PAR D(b) PAR Wd BAR T(395) PAR T(354) BAR W,dVW

PAR

bound

CAMP on Mutant (fmol

and Wild-type

Surface

BAR

per well)

Control

+ IS0

% Seq.

Control

+ dbcAMP

% Seq.

2.8 32.0 33.0 3.3 9.7 2.9 3.6 6.1 4.7 4.3 3.3

0.6 9.9 9.4 3.2 2.7 0.8 1.0 1.6 0.9 1.1 0.6

78 69 72 3 72 72 72 74 81 74 82

2.8 ND ND 10.6 ND ND ND 5.1 ND 5.4 1.9

2.2 ND ND 8.2 ND ND ND 3.8 ND 3.8 1.2

21 ND ND 23 ND ND ND 25 ND 30 37

Cells expressing each of the mutants described in Figure 1 were exposed to 10-s M isoproterenol or 10e3 M dibutyryl CAMP for 30 min at 37°C as indicated. The concentration of surface BAR was measured by [sH]CGP binding as described in Figure 3. ND, not determined; control, untreated cells: + iso. cells treated with isoproterenol; + dbcAMP, cells treated with dibutyrul CAMP; % seq., the percentage of cell surface binding sites lost upon either isoproterenol or dibutyryl CAMP treatment. Data are the means of duplicate determinations, representative of 2-5 similar experiments.

nofluorescence with the monoclonal antibody (Figure 48). The specific immunofluorescence on this cell line was brighter than that of the cells expressing the wild-type protein, reflecting the P-fold higher expression of the mutant than of the wild-type 8AR (see Table 1.) The specific immunofluorescence of the cells expressing D(239-272) PAR was not decreased upon treatment with isoproterenol, correlating with the [3H]CGP binding results shown in Figure 3 and contrasting with the results for the wild-type protein. No specific immunofluorescence of control cells not expressing the f3AR was seen with the monoclonal antibody (Figure 4C), which is consistent with the lack of detectable 8-adrenergic ligand binding sites. Functional Desensitization of Receptors To investigate the functional consequences of the sequestration of the 8AR binding sites, membranes were prepared from cells that had been exposed to isoproterenol, allowing both ligand binding and adenylate cyclase activity to be determined. As shown in Figure 5, isoproterenol-induced down-regulation of the PAR was reflected in the membrane preparation as a time-dependent loss of 1251-CYP binding activity. Approximately 50% of the receptor sites were lost from the membrane preparation in 1 hr, with a maximal down-regulation of 80% after 16 hr. This down-regulation of 8AR number was accompanied by a similar loss of isoproterenol-stimulated adenylate cyclase activity. There was no corresponding loss of NaF-stimulated adenylate cyclase activity upon treatment of the cells with isoproterenol, indicating that homologous desensitization was taking place in these cells. Isoproterenol-mediated adenylate cyclase stimulation by wild-type and mutant 8AR was measured in membranes prepared from untreated cells and from cells that had been exposed to isoproterenol, with the results shown in Table 3. Mutants D(k,z) BAR, T(354) BAR, and D(k,z)T(395) BAR all showed a marked decrease in isoproterenol-stimulated adenylate cyclase within 3 hr of exposure to isoproterenol and an almost total loss of this

activity after 16 hr. As with the wild-type BAR (Figure 5) the desensitization of adenylate cyclase activity in the mutants correlated well with the loss of ligand binding activity from the membranes (Figure 5). Discussion In this study, cells expressing the wild-type BAR responded to adrenergic agonists by rapid sequestration of the 8AR away from the cell surface, with the loss of 600/o-80% of the cell surface receptors within the first 30 min. Desensitization in some systems has been found to involve at least two separate processes, an uncoupling of the ligand binding activity of the 8AR from the cyclase stimulation, followed by a sequestration of the receptor away from the surface of the cell (Su et al., 1980; Waldo et al., 1983). This separation of desensitization from sequestration and down-regulation is not observed in all cell lines (Perkins, 1983). In the L cells used in the present study, no resolution of the time courses of these two events was detected and the down-regulation was sufficient to account for the desensitization observed. As has been observed with other cells, the decrease in SAR binding sites appeared to occur more rapidly when cell surface receptors were measured (sequestration, Figure 3A) than when binding to membranes prepared from the same cells was assayed (down-regulation, Figure 5). This suggests that some of the BAR initially lost from the cell surface were sequestered within the cell for a period of time before the ligand binding activity was completely lost from the system. After a 1 hr exposure to the agonist, however, the majority of the BAR binding sites were not detectable either at the surface or in the membrane preparation. This was confirmed by immunofluorescence experiments using an antibody that recognizes the PAR. Only the mutant D(239-272) BAR, which did not couple normally to the GJadenylate cyclase system, showed a reduced sequestration response to isoproterenol. This defect in sequestration was apparent with both ligand bind-

Cell 660

Figure

4. lmmunofluorescence

of DAR following

lsoproterenol

Treatment

Monolayers of cells expressing (A) BAR, (6) D(239-272) PAR, or(C) control cells were washed in PBS, then fixed in 3.7% formalin, I% ethyldimethyldiaminopropyl carbodiimide in PBS for 10 min at 23%. After permeabilization in 0.1% Triton X-100 for 5 min, cell monolayers were incubated with 4 mglml goat gamma globulin for 1 hr at 23% to decrease nonspecific binding of the antibody (Willingham et al., 1976). Cells were further washed with PBS, then incubated in PBS containing either normal mouse IgM (top panels) or purified monoclonal antibody fMR(226-239)3-l (middle and lower panels) at an antibody concentration of 0.1 mg/ml for 1 hr at 23%. After washing in PBS, the monolayer was incubated with FlTC-conjugated goat anti-mouse IgG (Cappel) at a 1:50 dilution for 1 hr at 23%. Cells were then washed extensively in PBS, and the fluorescence was observed using a Leitz Diaplan fluorescence microscope and photographed with a Leitz vario-orthomat camera system on the manual setting. All photographs shown are from the same experiment and were photographed under exactly the same conditions, with an exposure time of 15 sec. Cells in the bottom panel of groups A @AR) and B (D(239-272) f3AR) were treated with 10e5 M isoproterenol for 1 hr at 37% in the growth medium before fixation of the monolayer. The control L cells not expressing the BAR have a slightly higher background fluorescence than the lines shown in (A) and (B), reflecting differences among the clonal cell lines. The experiment shown is representative of 3 similar experiments.

ing activity and immunofluorescence. Cell lines expressing f3AR having smaller deletion mutations within this same hydrophilic region of the receptor did not display the defect in cyclase stimulation shown by 0(239-272) PAR (Table 1). These mutants, which coupled normally to G,, also showed a normal sequestration response to isoproterenol (Table 2), indicating that only the mutation which perturbed the coupling of the f3AR to the G-protein caused an altered sequestration response. Cells expressing D(239-272) PAR showed a desensitization response to PGE indistinguishable from that of control cells transfected with pSVL or cells expressing the wild-type 6AR.

Adenylate cyclase stimulation by PGE fell 500/o-80% after 3 hr with PGE in all three cell lines (data not shown), demonstrating that the mechanism of homologous desensitization was intact in cells expressing D(239-272) PAR. Earlier studies approached the issue of the involvement of G, in the sequestration and down-regulation of the BAR in the cyc- mutant of S49 cells, which lack a functional G, entity, and have given ambiguous results in that some studies found normal down-regulation in these cells (Green and Clark, 1981; Kassis and Sullivan, 1986) while others found the down-regulation to be defective (Shear et al., 1976; Su et al., 1980). One such study indicates that

~~Adrenergic 861

Receptor

Desensitization

0

2

tme

Figure 5. Time Course Wild-Type 8AR

wilh

16

3

tsoproterenoi

of Desensitization

denced by the normal cyclase stimulation and sequestration responses observed with mutants D(229-236) PAR, D(238-251) PAR, D(250-259) PAR, and D(k,) PAR. Thus, it appears that the sequence of the j3AR responsible for both the coupling to G, and the sequestration response can be localized to residues 263-272 of the mammalian &AR. The simplest explanation for the correlation observed between G, activation and receptor sequestration would be that G, is directly involved in f3AR sequestration. Alternatively, this region of the receptor could also contain the binding site for another protein that might be involved in BAR desensitization in a manner analogous to that in which arrestin interacts with rh~opsin (Wilden et al., 1986). Still another explanation would be that this sequence of the BAR is not directly involved in the interactions that accompany activation or sequestration, but that a large deletion in this region imposes conformational constraints on the j%AR that impair another function, such as aggr~ation, that might be involved in both G-protein coupling and sequestration of the receptor. In contrast to the attenuated sequestration observed with D(239-272) PAR, mutant receptors lacking either the C-terminus, the putative PKA sites, or both regions were sequestered normally in response to isoproterenol. We explored the role of camp-dependent phosphorylation on the observed sequestration of the BAR by exposing cells to either PGE, 8-bromo CAMP, or dibutyryl CAMP While no alteration in the number or activity of ceil surface receptors occurred with PGE or 8-bromo CAMP, a small decrease in cell surface sites was observed with millimolar levels of dibutyryl CAMP Interestingly, the same low level of sequestration induced by dibutyryl CAMP was observed for all of the mutants. Either this dibutyryl CAMP effect was indirect, involving phosphorylation of other membrane proteins, or residues on the 8AR other than those deleted in these mutants serve as sites for phosphorylation by PKA. The small response observed for dibu~ryl CAMP and the lack of a response for either 8-bromo CAMP or PGE indicate that the major portion of the sequestration observed with agonist proceeded through a non-CAMPmediated mechanism. Because of the rapid and almost complete sequestra-

(hr) and Down-Regulation

of the

L cells expressing the 8AR were treated in monolayer culture with 10-s M isoproterenol under normal growth conditions at TPC for the times indicated on the x-axis. Cells were then washed and membranes were prepared as described in Experimental Procedures. Memb~nes were assayed for ‘*sl-CYP binding activity (open triangles) or for isoproterenol (filled circles)- or NaF (open circles)-stimulated adenylate cyclase activity as described in Table 1. Data shown are the means of 5 separate experiments, with each determination performed in triplicate. Maximal binding of ‘ael-CYP ranged from 50-100 fmollmg protein. Basal cyclase activity ranged from 7-10 pmol cAMP/mg/min, with the NaF stimulation ranging from 22-34 pmol cAMP/mg/min among the 5 experiments.

G, may be required for long-term down-regulation but not for rapid sequestration (Mahan et al., 1985). Our present study suggests a correlation between the coupling of the j3AR with the GJcyclase system and its ability to undergo agonist-mediated sequestration. These processes both require the presence of residues in the sixth hydrophilic segment of the BAR. Neither the interaction with G, nor the sequestration of the receptor involves the entire sequence of the sixth hydrophilic loop of the PAR, as is evi-

Table 3. Desensitization

of Mutant

and Wild-Type lsoproterenol

PAA by lsoproterenol

Treatment -.

(Time)

0 Mutant Wild-type

PAR

D(b) PAR T(354) PAR WWWW

DAR

~~-~3 hr

.-....

.~~~

16 hr

ii-

I

F-

I3

I

F-

B

I

F.

9 11 12 7

31 44 29 17

30 35 24 21

7 6 5 4

12 15 8 6

31 31 18 18

7 8 7 8

9 13 9 8

36 31 24 20

Monolayers of cells expressing either BAR or mutant BAR were left untreated or were incubated with 10-s M isoproterenol for 3 hr or 16 hr under normal growth conditions as described in Figure 3. Cell membranes were isolated, and their adenylate cyclase activity was measured as described in Table 1. Adenylate cyclase activity is expressed here as pmol [s*P]cAMP formed/mg proteinlmin. B indicates basal activity; I, activity stimulated by 10m4 M isoproterenol; F-, activity stimulated by 10 mM NaF. The down-regulation of ligand binding sites (defined with 1251-CYP binding to the membranes) for the samples was as follows (% down-regulation after a 3 hr exposure to isoproterenol/% down-regulation after a 16 hr exposure to isoproterenol): BAR (70%/95%), D(krs) BAR (64%/89%); T(364) BAR (78%180%), D(krs)T(395) 8AR (49%/87%). Values given are the meane of at least two experiments, with each determination performed in triplicate. ND, not determined.

Cell 862

tion and down-regulation of the PAR in these ceils, it was not possible to isolate a population of desensitized PAR from this system, precluding the direct determination of the phosphorylation state of uncoupled receptors. One intriguing possibility is that, although sequestration proceeds normally in the PAR mutants lacking the serine-and threonine-rich C-terminus, phosphorylation of the C-terminus of the wild-type PAR might be required for its desensitization. According to this hypothesis, the C-terminus would exert an inhibitory effect that would be removed by agonist-induced phosphorylation. By analogy with the desensitization of rhodopsin, such an inhibition might involve the binding of an ancillary protein such as the 48 kd S-antigen, arrestin. Accordingly, the lack of normal sequestration observed with D(239-272) PAR could reflect the involvement of this region of the PAR in such a protein interaction. Although, these L cells contain measurable levels of PKA activity (data not shown), the presence of BARK in this system has not been demonstrated. However, in these cells, the desensitization can be completely accounted for by the sequestration and down-regulation of the PAR, which does not require the presence of either the C-terminal portion of the receptor or the putative PKA sites, but which does require the presence of a region involved in the interaction with G,. Future studies using single amino acid substitutions and cells in which the desensitization and down-regulation processes can be distinguished functionally will be necessary to explore the involvement of these regions in other aspects of the desensitization process. Experimental

Procedures

Mutagenesis

and Expression

Recombinant DNA procedures were performed according to Maniatis et al. (1982). The nucleotide sequence for the hamster gene has been published (Dixon et al., 1986). The expression vector for the hamster BAR, pSVwAR (referred to as pSVHAM in Dixon et al., 1987), was derived by inserting the hamster PAR gene into the SV40-derived expression vector pSVL. Expression of the BAR in mammalian cells from pSV PAR is driven by the SV40 early promoter. RNA processing utilizes the splice signals Sto the coding region and the poly(A) addition site from the PAR gene. Oligonucleotide-directed mutagenesis by previously described procedures was utilized to introduce nucleotide substitutions (Gibbs et al., 1984; Dixon et al., 1987). The nucleotide sequences of the mutant plasmids were confirmed by dideoxy sequencing (Sanger et al., 1977). L cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum in a 5% CO2 atmosphere at 37°C. Transfections were performed as described (Dixon et al., 1987). Membrane Preparation and Receptor Assays L cell membranes were prepared by lysis of cell monolayers in 1 mM Tris (pH 7.5) for 15 min. Cells were then scraped from the flask and centrifuged at 40,000 x g for 15 min. The pellet was resuspended at 1 mglml in TME buffer (75 mM Tris [pH 7.51, 12.5 mM MgCIP, 1.5 mM EDTA). Ligand binding activity was measured as described in Table 1 and Figure 3, by the methods of Mukherjee et al. (1976). Adenylate cyclase stimulation was determined as previously described (Dixon et al., 1987). Briefly, 20 ~1 of the membrane preparation containing lo-50 pg of membrane protein was mixed with 30 pl of a solution containing 7 mM phospho(enol) pyruvate, 0.3 mM ATP (including lo6 cpm ]@‘P]ATP), 0.3 mM CAMP, 0.1 mM GTP, 50 U/ml of myokinase, and 10 U/ml of pyruvate kinase. The mixture was allowed to react for 30 min at 30°C, then stopped at 4OC by mixing with an excess

of unlabeled ATP. The [32P]cAMP level was determined by the method of Salomon et al. (1974). For desensitization and sequestration experiments, L cells growing in monolayer culture were incubated under normal growth conditions either with 10m5 M (-)isoproterenol (Sigma) and 0.2 mM sodium metabisulfite lo enhance the stability of the agonist, or with 1 mM dibutyryl CAMP (Sigma), for the times indicated in the Figures. Monolayers were washed 3 times with PBS before additional assays were performed on either the intact cells or on membranes prepared from the cells, as indicated. Antibody Production The synthetic peptides CLDSQGRN(Nle)STNDSPL (residues 404-418 of the hamster BAR) and YAKRQLQKIDKSEGR (residues 226-239 of the hamster PAR) were synthesized and coupled lo thyroglobulin for use as an antigen as described previously (Dixon et al., 1987). The peptide QVAKRQLQKIDKSEGRFHS (residues 224-242 of the hamster PAR) was produced as a ras fusion protein, as described (Dixon et al., 1986), which was used directly as an antigen. The antibodies to pep tides 224-242 and 408-418 were produced in rabbits and have been described previously (Dixon et al., 1986, 1987). The antibody to peptide 226-239 is a monoclonal antibody of the IgM class, designated BAR(226-239)3-l. This antibody was selected partially on the basis of immunofluorescent recognition of the BAR, and its specificity was confirmed by immunoprecipitation of 1251-CYP-labeled BAR (Zemcik and Strader, unpublished results). The antibody or nonimmune IgM was purified from ascites by DEAE-HPLC and was used at a protein concentration of 0.1 mglml. Details of the immunofluorescence experiments are given in Figure 4. lmmunopreclpitatlon of the BAR Membranes were prepared from L cell lines expressing the various BAR proteins as described above, resuspended in TME buffer at a protein concentration of l-3 mglml, and labeled with 500 pM 1251-CYP for 3 hr at 4OC. The membranes were washed and solubilized in 1.5% digitonin in 10 mM Tris-HCI (pH 7.2), 0.1 M NaCl (Tris-NaCI buffer) for 2 hr at 4’X, and the undissolved membrane pellet was removed by centrifugation at 160,000 x g for 15 min. The free 1251-CYP was removed from the supernatant by filtration on Sephadex G-50, and the 1*51-CYP-labeled DAR was immunoprecipitated from the supernatant as described in Figure 2. Acknowledgments We would like to thank Mr. R. Mumford and Ms. J. D’Alonzo for synthesis of the peptide antigens, and Mr. V. Strout for help with antibody production. We are grateful to Drs. E. M. Scolnick, E. H. Cordes, E. E. Slater, and R. J. Gerety for their support and for helpful discussions throughout this study. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisemenf” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

February

17, 1987; revised

April 6. 1987.

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