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187,

No.

September

3, 1992

30,

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AND

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1992

Pages

IDENTIFICATION OF AMINO ACID RECEPTOR FOR LIGAND BINDING

1426-1431

RESIDUES OF RAT ANGIOTENSIN BY SITE DIRECTED MUTAGENESIS

II

Yoshiaki Yamano, Kenji Ohyama, Shigeyuki Chaki, Deng-Fu Guo and Tadashi Inagami’ Department

Received

August

of Biochemistry, Vanderbilt Nashville, TN 17,

University 37232

School of Medicine,

1992

To determine the specific mechanism of l&and binding to angiotensin (Ang II) receptor AT, , mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to eptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys Y99 to Gln in the intramembrane domain strongly reduced the affinity to both [‘25I] Ang II and [‘251]-‘Sar, ‘Ile-An5 II whereas mutation of two other Lys had little effect, indicating involvement of Lys19 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in Nglycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies. 4 1992~~~~~~~~ pTe55,inc.

In plasma membrane the outer membrane transmitted

spanning receptors, it is believed that the ligand binds to

domain

and generates

signals in the cytosolic

in various forms to elicit intracellular

which hormone binding

is transduced

responses.

Molecular

to the cytosolic biochemical

regions

to be

mechanism by

signals has not been

clarified. A receptor transmembrane of G-proteins. receptor

mechanism

cloned

recently

(1 - 3) to be of the seven-

domain type whose signal is transmitted

by the release of the a subunit

Recent findings that the binding site for epinephrine

(another

intramembrane

possibly

for Ang II (AT,)

seven

domain

transmembrane

provide

a valuable

of seven transmembrane

has multiple

binding

domain

clue which

is

located

in

*To whom correspondence should be addressed.

1426

the

may lead to the activation

receptors (4). As Ang II is a peptide hormone

sites in its receptor,

0006-291 X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in arry fbrm reserved.

receptor)

in the l3,-adrenergic

and

Ang II may find its way into a

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1992

intramembrane

AND

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area of the receptor for its productive

least a part of the peptide and its non-peptide in the intramembrane The finding

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binding.

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It is still possible that at

antagonist may seek a binding site located

region. that the carboxy

essential for the binding

terminal

residues of Ang II or its analogs are

suggests the importance

of a positively

charged group in the

receptor (5). AT, receptor is particularly a critical role played by disulfide conformation

sensitive to reduction by dithiothreitol, bridges in the formation

which suggest

and maintenance

of or

of the receptor (5).

As we have cloned cDNA for Ang II and expression system has become available we have applied extracellular

site directed mutagenesis

domains and basic groups in the intramembrane

glycosyl group at potential

Materials

for testing the importance

N-glycosylation

domains.

of cysteines in Possible role of

sites was also examined.

and Methods

COS-7 cells (American Type Culture Collection) were cultured in Cell culture: Dulbecco’s modified Eagle’s Medium containing 10% fetal bovine serum (DMFWFBS), penicillin and streptomycin in 5% CO,/95% air at 37°C. Site-directed mutagenesis of rat angiotensin II receptor: The entire coding region of rat kidney angiotensin II receptor (open reading frame of 359 amino acid residues) was cloned into an E&U site of a plasmid pUC19. A KpnI-EcoRI fragment of 2 kb in size was subcloned into polylinker sites of a plasmid vector Bluescript II KS+, and single stranded DNA was prepared using helper phage R408. Site directed mutagenesis was performed following the procedure of Kunkel(6). Sites of mutations are shown in Fig. 1. The changed DNA sequences were confirmed by Sanger’s dideoxynucleotide

K-l

Figure 1:

Mutations of rat Ang II receptor. Cysteine mutants (C-l through asparagine mutants 3) are shown The asterisk

(N-l through

with the mutated shown

N-3) and lysine mutants position

in the K-3 mutant

that position. 1427

and changed

(K-l through

amino

means a stop codon

C-4), K-

acid residue. introduced

at

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1992

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sequencing method (7). Those mutant DNA fragments were excised from vector Bluescript II KS+ using restriction enzymes BarnHI and XhoI and introduced into an expression vector pcDNA1, a shuttle vector between mammalian cells and bacteria. Expression of mutant angiotensin II receptors and I&and binding analysis: The cloned mutant DNAs were transfected into COS-7 cells, which were suspended in phosphate buffered saline (PBS) at a density of 6.0 X 106 cells/ml. The cell suspension (300$) was mixed with 6pg of mutant DNA. The mixture was electroporated at 150 V, 250 PF in a 0.2 cm gap cuvette in a Gene Pulser Apparatus (Bio-Rad) (8). The cell suspension was mixed with 6 ml of DMEM/FBS medium and was divided into six wells of a 24 well microtiter plate. Three days after the transfection, binding analysis was performed following the procedure reported previously (9). [rzI]-Ang II and [lzI]-lSar, ‘Ile-Ang II were used as ligands, concentration of which was varied from 10 nM to 0.3 nM. [3Hjlosartan was also used over the same concentration range.

Results and Discussion The transient expression method, using rat Ang II AT, cDNA inserted in pcDNA1, produced

COS-7 cells with remarkably

f 0.47 fmol/

reproducible

levels of receptor site (Bmux=9.09

5 x 16 cells as wild type receptor) (Table 1).

1: [‘“I]-Angiotensin Il binding to wild type and mutant receptors expressed in COS-7 cells (the results of triplicate experiments). C, N and K indicate mutation of cysteine, asparagine and lysine to glycine, aspartic acid and glutamine, respectively, except for K-3 mutant in which a stop codon (*) was introduced at the position 310 to delate the cytoplasmic C terminus. Table

All mutants

Kd (nM)

wt

1.75 +

Bmax (fmo1/5X105 cells) 0.09

9.09kO.47

C-l ( 18-G)

24.18 f

1.93

C-2 (101-G)

17.01 f

1.03

9.60 +

0.84

C-3 (18OdG)

14.98 2

0.77

8.52

+

0.29

C-4 (274-G)

14.93 f

0.85

10.09

t

0.61

( 4-D)

1.92 +

0.16

7.29 f

0.18

N-2 (176-D)

2.33 +

0.19

8.73 _+ 0.32

N-3 (188-D)

1.75 f

0.26

6.05 +

0.29

K-l (58,6O-*Q)

5.36 +

0.43

7.82 f

0.30

1.86

8.84 2

0.76

0.11

8.21 f

0.19

N-l

K-2 (199-Q) K-3 (309-+*) Data represents analysis at 4-C.

13.44 f 2.72 _+

11.88

_+ 0.65

4 independent series of binding isotherms followed by Scatchard Results of the Scatchard analyses are presented as means + S.E.

1428

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AND

I 4

2

bound

Figure

2:

Scatchard mutants increasing

[‘?I

binding

6

Ang

in transmembrane

II (fmol/5

concentration

region,

K-l

of [‘=I]-Ang cDNA

analysis (Fig. 2) showed

in K-2 (lacking the positive charge

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x

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I 10

8

analysis of specific [‘251]-AngII

(0), K-l (0) and K-2 (*)

Scatchard

BIOPHYSICAL

1 O’cells)

binding and K-2.

to wild

type and lysine

Specific

bindings

II to COS-7 cells expressing

with

wild type

were determined.

marked of LyslW

(S-fold) reduction

in [‘251]Ang II

in the fifth transmembrane

whereas in K-l (lacking Lysss and Lys60 in the first cytosolic loop), marginal the ligand affinity was observed. Interestingly,

column), change

the binding of [1251]-1Sar,‘-Ile-Ang

in

II was

weakened to an even greater extent (30-fold) in K-2 (Table 2). These results indicate that Ly?

in the fifth transmembrane

Table

domain provides an important

positively

2: [‘251]-‘Sar, ‘%e-AngII binding to wild type and mutant expreSsed in COS-7 cells AII mutants

wt

1.85 f

receptors

Bmax @mole/ 5X16 cells)

Kd (nM) 0.39

15.37

f

1.51

K-l

19.62

f

2.09

14.65

2

1.85

K-2

61.58

+

5.93

12.32 f

1.19

K-l and K-2 mutants used in thii experiment are the same as those used in Table 1. Results were obtained from 4 series of binding isotherms at 4°C followed by Scatchard analyses, and expressed as means + SE.

1429

charged site

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for the binding particularly

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of carboxyl

since the binding

AND

terminal

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carboxylate

of ‘Sar, ‘Ile-Ang

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anion of Ang II or its analogs,

II, lacking the amino terminal

residue is more extensively weakened than Ang II (10). While the mutation Gln did not result in the complete inactivation a peptide receptor in which binding total binding contention

affinity

sites may be distributed

may be contributed

Mutations

to several regions and the

from these sites. Partial support

to the above

in K-3 in which

which may drastically change the overall configuration

of the receptor

compounds

deleted.

receptor for ligand binding, mutated

Ang II receptor has been noted for its sensitivity

such as dithiothreitol

bridges in the maintenance

individually

with

affinity

may also affect the receptor function. to sulfhydryl

of LYS’~~to

it is not unexpected

may derive from the absence of effect on the binding

carboxy terminal tail is completely

disulfide

of the binding,

aspartyl

(5).

or formation

of the functional

to Gly.

conformation

of

of the

domains were

As shown in Table 1, C-l (C18G), C-2 (ClOlG),

C-3

10 fold increase in K,, whereas Bmax

Ang II receptor C”’ and C18’ in rat are highly

with seven transmembrane

the importance

cysteine residues in the outer membrane

(C180G), and C-4 (C274G) showed approximately were unchanged.

To examine

conserved compared

receptors (11-13) and are believed to form a disulfide bridge.

The other set of cysteine residues, Cl8 and C274 in rat Ang II receptor, is found in a few other peptide receptors (14,15). Although disulfide bridge is yet forthcoming, seems to be, at least partially, Thus, two disulfide domain

no definitive

the well noted sensitivity

due to the destruction

of AT, to dithiothreitol(5)

of these unique disulfide

bridges.

bridges formed by these pairs of Cys residues in the extracellular

seem to be essential for the receptor

octapeptide

chemical evidence for the two

configuration

for accommodating

the

Ang II.

Interesting is the finding that all of the mutations the non-peptidic

ligand [3H]-losartan

the nonpeptidic

ligand is less stringent

of Cys to Gly show no effect on

which suggests that the structural compared

requirement

with that for the peptide

for

(data not

shown). AT, has 3 potential Asn-X-Ser/Thr moiety

in its putative extracellular

is not understood,

localization individual

N-glycosylation

, transport, replacement

which presumably

domain. While the import of the N-glycosyl

the carbohydrate

stability

chains could contribute

and even ligand binding

to the surface

of the receptor.

of the three Asn by Asp (N-l: N4D, N-2: Nl76D,

eliminated

effect on Ang II binding,

sites with general consensus sequences of

Effects of

N-3: N188D),

glycosyl moiety at each of the Asn residue, showed little

just the same result as m2 muscarmic

(16). 1430

acetylcholine

receptor

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1992

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Thus, any one of the N-glycosyl

AND

BIOPHYSICAL

are comparable In summary,

functional

of

and the Bmax values of the

with that of the wild type (Table 1).

the site directed

mutagenesis

studies indicated

groups, two disulfide bridges in the extracellular

on the fifth transmembrane have significant

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moiety does not seem to affect the transport

the receptor to the outer surface of the plasma membrane mutants

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contribution

domain, which is presumably to the ligand binding

to AT,

that of various

domain and LYS~~~,located a hydrophobic

environment,

receptor.

Acknowledgments We would like to thank Ms. T. Fitzgerald and Ms. T. Stack for their technical and secretarial assistance. We also thank DuPont-Merck Company for providing us with Dup 753. This work was supported by Research Grants HLl4192 and HL32353 from the National Institutes of Health.

References

4. 5.

6. 7. 8. 9. 10. 11.

12.

13. 14. 15. 16.

Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ,Hasegawa M, Matsuda Y and Inagami T: Nature, 351, 230-233, 1991. Murphy TJ, Alexander RW, Griedling KK, Runge MS and Bernstein KE: Nature, 351, 233-236, 1991. Iwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y and Inagami T: Biochem. Biophys. Res. Commun., 177, 299-304, 1991. Strader CD, SigaI IS, Candelore MR, Rands E, Hill WS and Dixon RAF: J. Biol. Chem. , X& 10267-10271,1988. Chiu AT, McCall DE, Nguyen TT, Carini DJ, Duncia JV, Herblin WF, Uyeda RT, Wong PC, WexIer RR, Johnson AL and Timermans PBMWM: Europ. J. Pharmacol., 170, 117-118, 1989. Kunkel TA: Proc. NatI. Acad. Sci. USA, 82-488-492, 1985. Sanger F, Nicklen S and Coulson AR: Proc. NatI. Acad. Sci. USA, Z& 5463-5467, 1977. Chu G, Hayakawa H and Berg I? Nucl. Acids Res., l5- 1311-1326,1987. Pandey KN, Pavlou SN and Inagami T: J. Biol. Chem., m 1340613413,1988. Khosla MC, Leese RA, Maloy WL, Ferreira AT, Smeby RK and Bumpus FM. J. Med. Chem., u 792795,1972. Kubo T, Fukuda K, Mikami A, Maeda A, Takahashi H, Mishina M, Haga T, Haga K, Ichiyama A, Kangawa K, Kojima M, Matsuo H, Hirose T and Numa S: Nature, 323, 411-416, 1986. Dixon RAF, Kobilka BK, Strader DJ, Benovic JL, DohIman HG, Frielle T, Bolanowski MA, Bennett CD, Rands E, Diehl RE, Mumford RA, Slater EE, SigaI IS, Caron MG, Lefkowitz RJ and Strader CD Nature, 321, 75-79,1986. Nathans J and Hogness DS: Cell, 34- 807-814,1983. Arai H, Hori S, Aramori I, Ohkubo H and Nakanishi S: Nature, 348, 730-732, 1990. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K and Masaki T: Nature, 348, 732735,199O. van Koppen CJ and Nathanson NM: J. Biol. Chem., 265, 28887-20892,199O.

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