Intrinsic photoaffinity labeling of native and recombinant rat pancreatic secretin receptors

Intrinsic Photoaff inity Labeling of Native and Recombinant Pancreatic Secretin Receptors CHARLES D. ULRICH APRIL CHANG-MILLER,

Rat

II, DELIA I. PINON, ELIZABETH M. HADAC, EILEEN L. HOLICKY, LAWRENCE K. GATES, and LAURENCE J. MILLER

Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota

BackRround: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for “intrinsic” photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. Methods: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. Results: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([1251]Tyr10,pN02-Phe22)rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. Conclusions: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonistbinding domain.

S

ecretin

is a 27-amino

acid peptide hormone

acinar cells,’ with its effects mediated

primarily

via

adenylate cyclase.6 Multiple binding sites for secretin have been identified in the pancreas. 7-9 These can be differentiated the basis of their affinities for structurally tides, with the secretin

on

related pep-

receptor

defined by its high

affinity for secretin and relatively

low affinity for va-

soactive

intestinal

structurally

polypeptide

characterize

pered by difficulties can efficiently

(VIP).‘**

in generating

covalently

Attempts

to

this receptor have been hamradioligands

that

label this receptor.

Affinity labeling is a powerful technique for the biochemical characterization valent labeling tional

of hormone receptors.”

may be achieved

chemical

photoactivatable

cross-linkers

or

monofunctional

ligands. ” To date, the only successful

attempt to affinity label the pancreatic tor used the cross-linking oxal, after [‘*‘I]secretin persed rat pancreatic band centered

was allowed to bind to disacini.‘*

This identified

by cold secretin;

use in structural characterization incorporating

however,

with

the effi-

low, limiting

its

studies.

a photolabile

group into a

domain 13-15have the additional theo-

retical advantage of labeling the ligand-binding of the receptor.

a broad

weight of 54,000,

ciency of this labeling was extremely Ligands

secretin recep-

reagent, p-azidophenylgly-

at a molecular

labeling inhibited

pharmacophoric

Co-

using either bifunc-

By introducing

relatively

region

small nitro

or azido moieties onto a pre-existing aryl residue, the resulting probe may continue to approximate the native conformation of secretin. Our group previously reported the use of pNO,-Phe-containing probes to

se-

creted by endocrine cells of the proximal small intestine. Since its discovery in 1902,’ secretin has been implicated in numerous physiological events, including roles in pancreatic exocrine and endocrine secretion.*y3 It is the princip al hormonal stimulant of bicarbonate and water secretion by pancreatic duct cells3p4 and contributes toward enzyme secretion by pancreatic

Abbreviations used in this paper: BSA, bovine serum albumin; HPLC, high-performance liquid chromatography; IC,, 50%~lnhibitory concentration; K,,, dissociation constant; KRH, Krebs’-RingerHEPESsolution with protease inhibitors; PCR,poiymerase chain reaction; PMSF, phenyimethylsuifonyl fluoride; SDS-PAGE, sodlum dodecyi sulfate-poiyacryiamide gel electrophoresls; STI, soybean trypsin inhlbitor. 0 1993 by the American Gastroenteroiogicai Association 0016~5085/93/$3.00

RECOMBINANT SECRETIN RECEPTOR

November 1993

characterize

Tyr’“,pN02-Phe22)rat

secretin

photoafhnity labeling approaches ceptor. I3 Intrinsic have also been used to characterize a number of other

logue).

were

and biologically.

To study the relationship

receptors.‘4*15 The primary

secretin-binding

protein

been

the binding

site of the cholecystokinin

and secondary

characterized.‘*‘“”

tionships

for this

various

hormone

secretin

though

critical

residues

that binding

and

critical

of the

to establish

agonist

velopment

would

on

photolabile macophore,

other

cretin-binding

protein

line (NG108-15)

that can be radioio-

than

His’,

incorporate

regions

secretin

of the pharaffinity

and

the molecular

ar-

was identified

derived

from

member

amino

Appropriate

binding

monophosphate

were observed

binding

in the

with whole

encodes

protein,

affinity

the broad

nature

family and

5’-cyclic to sethis reanalysis

was positive

of reduced

pancreas.

in

inten-

Although

a G protein-coupled and studies

of

it as a

hybridization organs

en-

of recep-

responses

a signal

tity with the major high affinity tein in rat pancreas

analysis

confirming

specificity

this recombinant

labeled,

the cDNA

in COS cells expressing

rat heart and stomach, clearly

cell

with

(CAMP)

combinant protein.28 Northern of poly(A)+ RNA from various sity detected

in a hybrid neuroblastoma

protein,

of the G protein-coupled

guanosine

a se-

mouse

acid sequence

have also

Recently,

et al. cloned

this secretin-binding

the predicted

receptor

preparations.

and rat glioma. 27 Ishihara

been

se-

aided by the de-

to characterize

by substrate

cDNA

is

appropriate

high binding

of the pancreatic

been limited

cretin

and

this

secretin-

receptor

has never

confirming

its iden-

secretin-binding

have yet to be performed.

of the competition-binding

probes

27 (pN02-Phe22 characterized

described

and the rat pancreatic

secretin

of a rat pancreatic

screening

chemically between

by Ishihara

bridization

receptor,

anathe

et a1.28

we used hylibrary

to

clone a full-length cDNA. Further, using the pNO,Phe22 analogue, we have successfully affinity labeled the major

receptor

secretin-binding

and have compared protein

in native

it with rat pan-

creas.

frag-

of the pancreatic

into differing

attempting

chitecture

tors.%

shown

region

These

this recombinant

its

activity.

Studies

coding

activity

and still retain

biological

have

amino-terminal

analogues

a residue residues

Al-

to exert

studies

be substantially

of secretin

dinated

using

analogues.2,2”-23

is necessary

22,26 Characterization

receptor

studied

using a carboxyl-terminal

addition

specificity.

has rela-

such as His’ have been identi-

molecule

is possible

but

cretin

been

effects. 2,20-23 Detailed

biological

of secretin

structure-activity have

fragments

fied 24*25the entire

ment,

structure

The

re-

1535

proIndeed,

curve ob-

served in native rat pancreatic cells suggests the possible presence of several secretin-binding proteins, as well as multiple affinity states of these proteins.7,s In an attempt to site a photolabile residue into a region of secretin, which would be in close apposition to the receptor yet still permit binding and biological activity, we have designed and synthesized two novel (pN02-Phe6,[‘251]Tyr’o,Glu’5, secretin analogues, Glyi6)rat secretin 27 (pNO,-Phe6 analogue) and ([‘251]-

Materials and Methods Synthetic rat secretin, porcine VIP, and cholecystokinin 8 (CCK-8) were purchased from Peninsula Laboratories (Belmont, CA). Protected amino acids and methylbenzhydrylamine resin were from Advanced ChemTech (Louisville, KY) and Peninsula Laboratories (Belmont, CA). All solvents were high-performance liquid chromatography (HPLC) grade, and all reagents were either analytical or molecular biology grade. Harlan Sprague-Dawley rats were used as source of pancreatic tissue. Protocols were reviewed and approved by the Mayo Clinic Animal Care and Use Committee.

Secretin Receptor Probe Development and Characterization Synthesis of peptides. Secretin analogues were synthesized manually, following the standard cycles of coupling and deblocking of resin we have previously reported.‘” The two 27-amino acid peptide analogues of secretin illustrated in Figure 1 were synthesized on p-methylbenzhydrylamine resin (1.5 g, 0.4-0.5 mmol). The resin was treated with HF (30 mL, 2 hours, -10°C) containing anisole (1 mL). After evaporation of the HF, the residue was washed with ether (100 mL), extracted with water (50 mL), and lyophilized. The crude product was purified by semipreparative reversed-phase HPLC on octadecylsilica (Vydac 218TPlOlO column; 1 X 25 cm; 10 pm C-18; pore size, 300 A; Nest Group, Southborough, MA), using a flow rate of 4 mL/min, with 0.1% trifluoroacetic acid and a linear acetonitrile gradient from 10% to 60% more than 50 minutes followed by a second separation using a linear acetonitrile gradient from 20% to 40% over 30 minutes. The synthetic analogues were characterized by analytical HPLC and quantitative amino acid analysis. For HPLC, we used a Vydac 218TP54 reversed-phase column, at a flow rate of 1 mL/min, with the same buffer system described. (pN0,-Phe6,Tyr’o,Glu15,Gly’6)rat secretin 27 (pNO,-Phe6 analogue) eluted as a single symmetrical peak at 37.9% acetonitrile, and (Tyr”,pNO,-Phe**)rat secretin 27 (pNO,-Phe*’ analogue) eluted as a single symmetrical peak at 37.5% acetonitrile on this system. Yields were 33.6% for the pNO,-Phe6 analogue and 42.6% for the pNO,-Phe** analogue. Peptide hydrolyses were performed in 6N HCl for 22 hours at 11O’C

1536 ULRICH ET AL.

GASTROENTEROLOGY Vol. 105, No. 5

Native peptides

Photolabile analogues pN02-Phes analogue

pNO2 @

@

pN02-Phe22 analogue

pNO2 @

0

Figure 1. Design of pNO,-Phe-containing rat secretin analogues. Shown are the amino acid sequences of native secretin molecules in rat, human, and chicken, as well as the sequences of secretin analogues used in this work. All peptides were 27 amino acids long, with only the residues that were different from rat secretin 27 depicted. Sitesfor pNO,-Phe (shaded) and Tyr (bold) substitution in the analogues were selected based on a comparison of the sequences of secretin across species. (pN0,-Phe6,[‘251]Tyr’0,Glu’5,Gly16)rat secretin 27 (pNO,-Phe6 analogue) and ([1251]Tyr10,pN02-Phe22)rat secretin 27 (pN02-Phe2* analogue) were both designed to provide high-specific radioactivity, high-affinity ligands that incorporate a photolabile residue into a pharmacophoric region, permitting “intrinsic” photoaffinity labeling.

in a sealed vessel under were performed (Beckman lithium

Instruments, buffer

acid analyses

cation

acid analyzer

cised pancreata

Fullerton,

system.

thetic peptides

Amino

7300 amino

high vacuum.

on a Beckman

CA) using the standard

The expected

were confirmed

identities

by their amino

Radioiodination of probes. Both dinated

with

Co., Rockford,

IL).”

(Iodo-Beads; Briefly,

in 90 PL of 0.2 mol/L

borate

incubation

was purified

dac C-18 column acetonitrile

at room a linear

gradient

in 0.1% trifluoroacetic

of 1% per minute.

The radioiodinated

containing

and 1 mmol/L homogenized

with

centrifuged rotor.

was overlaid

for 3 hours

Membranes

HPLC

using a Vy-

Krebs’-Ringer-HEPES

from

10% to 60%

(KRH)

was separated

washed,

(25 mmol/L

mmol/L

KCl,

mmol/L

KH,PO,,

1.2 mmol/L 0.2%

0.01% STI, and 1 mmol/L

by reversed-phase

says, membranes

specific

radioactivity.26

Biological activity of probes. The biological

activi-

ties of both pNO,-Phe-containing analogues were determined by measuring their abilities to stimulate amylase release from dispersed rat pancreatic acini. Acini were prepared from male Harlan Sprague-Dawley rats (125-l 50 g body weight) by sequential enzymatic and mechanical dissociation.31 Amylase release assays were then performed with varying concentrations of unlabeled analogue using the Phadebas reagent.32,33

Characterization of Native Rat Pancreatic Secretin Receptor Membrane preparation from rat pancreas. Enriched pancreatic plasma membranes were prepared from similar rats to those used to assess biological activity, using a modifi-

with pestle B. The sam-

with

bovine

were incubated association cubations

in KRH.

interface

protease

inhibitors

2 mmol/L

serum

and

bucket

in a modified

pH 7.4, 104 mmol/L MgSO,,

was

sucrose.

sucrose

mol/L

at -70°C

NaCl, 5 CaCl,,

albumin

1

(BSA),

PMSF).

Receptor binding studies. In standard (l-10

sucrose

to 1.3 mol/L

to the 0.3-1.3

solution

and

4 strokes

with 0.3 mol/L

HEPES,

(STI)

(PMSF),

2.0 mol/L

and stored

from native peptide, to yield specific radioactivity of 2000 Ci/mmol. VIP was oxidatively radioiodinated and purified HPLC to a similar

inhibitor

at 149,OOOg in a swinging

floating

were collected,

product

and enough

ex-

in 0.3 mob

homogenizer,

added to bring the final concentration This homogenate

In brief,

fluoride

by 4 strokes

filtered

the reaction

at a rate

trypsin

a glass dounce

with pestle A followed

was added. After

acid, increasing

0.01% soybean

ple was then

Chemical

described.34

up to 10% wt/vol

phenylmethylsulfonyl

io-

pH 9.0, con-

temperature,

L sucrose,

previously

were brought

N-

was dissolved

buffer,

by reversed-phase

with

were oxidant

Pierce

Na’*‘I (1 mCi), and one Iodo-Bead

a 15-second mixture

peptides

10 ktg of peptide

sodium

syn-

acid analyses.

Nalz51 by use of the solid phase

chlorobenzenesulfonamide

taining

of both

of the method

pg) and Time-

3-5

pmol/L

binding

as-

radioligand

and temperature-dependent

experiments were performed using 500~/,tL inat 4’C, room temperature, and 37°C. Aliquots

were taken in duplicate at specified times and diluted to 1 mL with KRH at 4”C, immediately centrifuged (15,OOOg for 5 minutes) to separate bound from free radioligand, washed, pelleted, and counted.34 Based on these findings, all subsequent binding studies were performed at steady state, attained after 10 minutes at 37°C for the pNO,-Phe** analogue, and after 60 minutes at room temperature for the pNO,-Phe6 analogue. Nonspecific binding of each radioligand was determined in the presence of excess unlabeled analogous peptide (1 ltmol/L secretin or VIP). Photoaffinity labeling. Photoaffinity labeling studies

RECOMBINANT

November 1993

were performed as we have described.‘3 In brief, initial binding of radiolabeled probe to membranes was performed as described above, except that more radioligand, 75-100 pmol/L, and more membrane protein (SO-100 pg) were used. The membranes were then pelleted by centrifugation, washed with 1 mL of KRH without BSA, pelleted by centrifugation, and resuspended in 1 mL of KRH without BSA. The labeled membranes were then transferred to 12 X 75mm borosilicate tubes for photolysis, which was performed for 30 minutes at 4’C with a Rayonet model RP-100 apparatus (Southern New England Ultraviolet, Hamden, CT) equipped with 300-nm lamps, the cooled samples separated from the lamp by 5.7 cm. Photolysis conditions for pNO,Phe-containing peptides were previously established in this lab to assure the specific activation of this residue.13 Membranes were collected by centrifugation and analyzed by 10% sodium dodecyl sulfate-polyacrimide gel electrophoresis (SDS-PAGE)35 followed by autoradiography. The molecular weights of affinity-labeled proteins were calculated from a plot of log of molecular weight vs. mobility of standard proteins. The range of these values reported represents the predominant region labeled in 6 independent experiments. Fifty-percent-inhibitory concentration (I&,) values for covalent labeling were determined using densitometric scanning of autoradiographs. Enzymatic deglycosylation. Affinity labeled membranes (So-100 pg) were prepared for deglycosylation by suspension in 50 pL of 0.1 mol/L sodium phosphate, pH 6.1, containing 50 mmol/L EDTA, 1% Nonidet P-40,0.1% SDS, and 1% 2-mercaptoethanol. Endoglycosidase F (endo F; 5 U) was added, and the incubation was allowed to proceed for 12 hours at 37°C. An equal volume of sample buffer was then added, and samples were analyzed by SDSPAGE and autoradiography, as described above.

RECEPTOR

1537

A 32P-labeled probe was generated by PCR using nested primers ii and iii and this cDNA fragment as template.36 In brief, PCR was performed as described abcve, with changes including only 50 pmol/L cold dCTP, the addition of 1.5 mmol/L [a-32P]dCTP (6000 Ci/mmol), and the use of the nested PCR product as template. cDNA library screening. Competent Escheri& cd’ (MC1061/P3) were transformed with the aforementioned rat pancreatic cDNA library and screened by hybridization with the 32P-labeled probe. Three and a half million colonies were initially screened, and potential positives were purified by another round of hybridization. Recombinant plasmids containing the target sequences were isolated and verified by restriction mapping and double-stranded DNA sequencing. Because the cDNA of interest was in the wrong orientation, it was subcloned into pcDNAl/Neo at the iWI and Hind111 sites. Recombinant receptor expression. The pcDNAl/ Neo clone was isolated by the alkaline lysis method, purified by centrifugation to equilibrium in cesium chloride-ethidium bromide gradients, and transfected into COS-7 cells using a modified DEAE-dextran protocol and CHO cells by lipofection.37 Recombinant receptor-bearing COS cells were harvested 48-72 hours posttransfection. Receptor-bearing cells were washed with PBS, scraped into a conical tube, and pelleted at 1000 rpm for 2 minutes. The cell pellet was resuspended in 0.3 mol/L sucrose containing 0.01% ST1 and 1 mmol/L PMSF and lysed with 5 strokes in a Potter-Elvehjem tissue homogenizer. The remainder of the preparation was identical to that described for rat pancreatic membranes. Binding and photoaffinity labeling studies using these membranes were performed according to the protocols described above.

Results

Pancreatic Secretin Receptor Cloning and Expression Polymerase chain reaction probe amplification. Four oligonucleotide primers corresponding to nucleotides 271-287 (i), 352-362 (ii), and complementary to 845-861 (iii), and 944-960 (iv) of the published cDNA sequence encoding a secretin-binding proteinz8 were synthesized. DNA amplification was performed using recombinant Taq DNA polymerase in a thermal cycler programmed to denature at 94°C for 1 minute, anneal at 52’C for 2 minutes, and extend at 72’C for 3 minutes. A custom-synthesized rat pancreatic cDNA library was used as a template for oligonucleotide primers i and iv. After 35 cycles, agarose gel electrophoresis showed a band of the predicted size. This band was then excised and reamplified using nested primers ii and iii under identical polymerase chain reaction (PCR) conditions. Subsequent agarose gel electrophoresis confirmed amplification of the predicted size cDNA. This cDNA was gel-purified, blunted using the Klenow fragment of DNA polymerase I, cloned into the SmaI site of MlSmpl9, and sequenced using the dideoxy chain termination method (sequenase v.2).

SECRETIN

Secretin Receptor Probe Development Characterization

and

Probe synthesis and chemical characterization. Each synthetic secretin analogue was purified by HPLC

to yield a sharp peak, with its structure

by amino

Biological activity. Amylase ing dispersed analogues

verified

acid analysis. rat pancreatic

were equally

tive rat secretin

(Figure

acini

efficacious

release

studies

showed

that

secretagogues

2). The analogues

usboth

to na-

were some-

what less potent than native secretin, however, with half-maximal secretion stimulated by 10 nmol/L secretin, nmol/L

50 nmol/L pNO,-Phe6

pNOa-Phe2* analogue.

analogue,

and

300

Characterization of Native Rat Pancreatic Secretin Receptor Binding characterization with rat pancreatic membranes. Both radioligands bound in a time- and

1538

ULRICH

Ok” 0

ET AL.

’ -11

GASTROENTEROLOGY

I

I

I

1

I

I

-10

-9

-0

-7

-6

-5

Peptide

concentration,

log M

Figure 2. Ability to stimulate amylase secretion from dispersed rat pancreatic acini. Various concentrations of the pNOz-Phe6 analogue (0), the pNO,-Phez2 analogue (I), and secretin (A) were incubated with acini for 30 minutes at 37°C. Maximal amylase secretion (expressed relative to maximal secretion stimulated by 0.2 nmol/L CCK8) was not statistically different for any of the three peptides (P < 0.05). The pNO,-Phe6 analogue was 30 times less potent and the pNO,-Phe** analogue 5 times less potent than native secretin. Each value represents the mean + SEM of three separate experiments performed in duplicate.

manner (Figure 3). Binding of temperature-dependent the pNOz-Phe6 analogue reached its maximal level after 60 minutes at room temperature, whereas binding of the pNO,-Phez2 analogue reached its maximum level after 10 minutes at 37°C. Both probes bound specifically and with high affin-

pNOn- Phe”Analogue

pN0~ - Phe’Analogue 100

Z” ._

m

No. 5

ity to pancreatic membranes. Nonspecific binding, determined in the presence of 1 pmol/L unlabeled secretin, represented less than 20% of total binding for the pNO,-Phe6 analogue and less than 22% for the pN02Phe22 analogue. In each case, specific binding was linearly related to the amount of membrane protein used in the incubation. Under the standard conditions used, 2% + 0.1% of the pNO,-Phe6 analogue and 14% + 1% of the pN02-Phe22 analogue were specifically bound at steady state. Competition binding studies showed inhibition of specific binding in a concentration-dependent manner by secretin, cold analogue, and VIP (Figure 4). In studies performed with the pNO,-Phe6 analogue, the concentration required to inhibit 50% of secretin, 3 specific binding (I&,) was 1 nmol/L nmol/L unlabeled analogue, and 0.3 /.kmol/L VIP. Using LIGAND (BCTIC, Nashville, TN), the calculated dissociation constant (Kd) for this analogue was 0.94 f 0.4 nmol/L. For the pN02-Phe22 analogue, the ICso values were 0.2 nmol/L secretin, 0.3 nmol/L unlabeled analogue, and greater than 1 pmol/L VIP. The calculated Kd for this analogue was 4.5 + 0.8 nmol/L. Using these Kd values for the radioligands, the calculated Kd values for native secretin were similar for both series of studies (0.56 f 0.2 nmol/L using the pNO,Phe6 analogue and 1.1 k 0.3 nmol/L using the pNO,Phe22 analogue; P = 0.11). These findings confirm the high affinities and specificity of both analogues for the rat pancreatic secretin receptor. To show the low affinity of these analogues for rat pancreatic VIP receptors, binding studies were performed with [‘251]VIP (Figure 5). In each case, analogue competition binding curves were similar to the

Lo

pN0~ _Phe=Analogue

pN9 - Phe”Analogue

5

$40

Vol. 105,

100

Lo 0

Time, min Figure 3. Time- and temperature-dependence for binding to native rat pancreatic membranes. In each panel, pancreatic membranes were incubated with a radioligand based on the noted secretin analogue at 37”C, room temperature, or 4°C. Aliquots were removed in duplicate at specified times, diluted to 1 mL with KRH at 4”C, and centrifuged to separate bound from free ligand. Specific binding of the radioligand is shown, expressed as a percentage of maximal for each experiment. (A) Binding of the radioiodinated pNO,-Phe’ analogue was more rapid at 37”C, but maximal at room temperature after 1 hour (mean of three experiments performed in duplicate). (6) Binding of the radioiodinated pN02-Phez2 analogue was both more rapid and maximal at 37”C, reaching its peak at 10 minutes (mean of two experiments performed in duplicate).

0 0

-11 -10 -9

-a

-7

4

-5

0

-10

-9

-5

-7

-6

Peptideconcentration,log M Figure 4. Competition for binding of radiolabeled secretin analogues to native pancreatic membranes. Membranes were incubated with a radioligand based on the noted secretin analogue plus varied concentrations of secretin, unlabeled secretin analogues, and VIP. 100% binding represents specific binding in the absence of competing peptide. Both analogues bound specifically and with high affinity to pancreatic membranes. Each value represents the mean + SEM of three separate experiments performed in duplicate.

November

RECOMBINANT

1993

cells.*’

100

This

mapping

B ha0

identity

SECRETIN

was

RECEPTOR

confirmed

and double-stranded

by

DNA

1539

restriction

sequencing.

8

Recombinant receptor expression and characterization. Expression of this secretin-binding protein

360 8 2 40

using

in transfected binding

a m20 o

-10

0

-9

-7

-9

Peptide concentration,

-6

log M

secretin

VIP,

0.2 pmol/L

pNO,-Phe6

curve,

with

I&,

pNO,-Phe**

analogue,

values

of 0.1 nmol/L

analogue,

and 0.4 ymol/L

analogue

cells.

Binding

branes

from with

previously 1

probes,

only

membranes logue.

was successful

Figure

6 shows

autoradiographs. 6B was

The experiment

performed

competing

with

cold secretin,

was performed

varying

a plasma

lar weight

of 50,000-62,000 inhibited

covalent

that

protein

in Figure

of 6C

of VIP. In

with a molecu-

was specifically labeling

binding

pN02-Phe22

labeled

in membranes

a

as documented was

secretin-binding

of secretin

of this protein,

secretin

abolished 2 nmol/L

F after affinity

had

weight

in the zymogen-rich

(mol

to yield of 42,000

trailing

wt,

deglycosylated the same (Figure

of deglycosylation

some

size. This is consistent

were

labeling

major band at a molecular

in the sizes of

receptor

of these

the product

this labeling.

(mol wt, 50,000-62,000)

secretin

both

covasecretin

and concentrations

differences

receptor

57,000-62,000),

receptor

with

VIP not inhibiting

recombinant

Of interest,

7). Increasing

binding

of the apparent

endo

this recombi-

(Figure

completely

50% of specific

the

for both successfully

expressing

protein

lent labeling

Because

affinities analogue

57,000-62,000-molecular

concentrations

tive

mem-

high-affinity

to smaller

apparent

with some proteolysis cell, likely during

8).

of the naoccurring

fractionation.

labeled.

of this protein

by

the pN02-Phe22 analogue in a concentration-dependent manner, with an IC,, of 4 nmol/L. VIP concentrations of up to 0.1 l.tmol/L lent labeling.

similar

the

protein

with

in Figure

concentrations

concentrations

membrane

ana-

SDS-PAGE

illustrated

although

with varying

all studies, Secretin

only with pNO,-Phe**

two representative

showed

specificities

untrans-

with

et al.*’ The IC,, for secretin

photoaffinity

and

plasma

or the

from

performed

cells

weight

the native

of rat pancreatic

analogue

to membranes studies

structural

despite

logues,

labeling

studies saturable

and for VIP was 1 l.tmol/L.

Again,

Photoaffinity labeling of pancreatic membranes. Despite similar binding affinities for both anaphotoaffinity

pNO,-Phe6

by Ishihara

nmol/L

by binding

was no specific

transfected

up to 1 pmol/L

secretin.

the

fected

inhibiting

0.2 pmol/L

There

pNO,-Phe**

nant native

probes.

of either

binding,

Figure 5. Competition for binding of [rz51]VIP to native pancreatic membranes. Membranes were incubated with radioligand plus varied concentrations of secretin, unlabeled pN02-Phez2 analogue, unlabeled pNO,-Phe’analogue, and VIP. For both unlabeled secretin analogues, competition binding curves were similar to the native secretin curve. Values represent the mean t SEM of three separate experiments performed in duplicate.

cells was confirmed

both

had no effect on its cova-

Pancreatic Secretin Receptor Cloning and Expression PCR probe amplification and cDNA library screening. Nested primer PCR using rat pancreatic pcDNA1 library as template produced a 509-base pair cDNA with 100% nucleotide sequence identity with the 5’ end of the cDNA reported by Ishihara et al.*’ 32P-labeling of this cDNA by PCR yielded a product with high specific radioactivity. Screening of a rat pancreatic pcDNA1 library identified one clone with a full-length insert that was identical to the cDNA encoding a secretin-binding protein found in NG10815

Attempts

to characterize

tecture

of the pancreatic

limited

by difficulties

gands and obtaining these limitations, ized two novel the

structure

the molecular

secretin in both

sufficient

receptor

generating substrate.

we have synthesized photoaffinity of

Tyr’“,Glu’5,Gly’6)rat

rat

labeling

secretin, secretin

archi-

have been suitable

li-

To overcome and character-

probes

based on

(pN02-Phe6,[‘251]27,

and

([1251]Tyr’0,

pNO,-Phe**)rat secretin 27. The amino acid substitutions required to permit both radioiodination and “intrinsic” photoaffinity labeling with these analogues were well tolerated, with both peptides maintaining full biological efficacy with high binding affinity. Only the pNO,-Phe** analogue successfully photoaffinity labeled a high affinity secretin-binding protein in rat pancreatic membranes. We then used these probes to characterize a recombinant rat pancreatic

1540

ULRICH ET AL.

GASTROENTEROLOGY Vol. 105. No. 5

[Secretin],

log M

[VIP], log M

0 -10 -9 -8 -7 -6

-10

-9

-8

-7

-6

-9 -8 -7 -6

29 -

[Secretin-271, log M

I3

A

Figure 6. Photoaffinity labeling of native pancreatic membranes by the radioiodinated pN02-Phe2* analogue. Membranes were incubated with radioligand in the presence of increasing concentrations of secretin and VIP, and then photolyzed for 30 minutes. The autoradiograms in (6) and (C) show that covalent labeling of the 50,000-62,000-molecular weight protein was competed for in a concentration-dependent manner by secretin and not inhibited by VIP. Inhibition of covalent labeling of this protein by secretin (A), depicted in (A), was quantified by densitometry. Specific labeling is expressed as a percentage of labeling in the absence of competing cold ligand. Four nanomoles per liter secretin inhibited 50% of specific labeling.

secretin

receptor,

confirming

its apparent

with the native rat pancreatic

(pN0,-Phe6,[‘251]Tyr’o,Glu15,Gly’6)rat and ([1251]Tyr’o,pN0a-Phe22)rat designed

to provide

high-affinity

radioactivity

functional

27

ficacious secretagogues. creatic

both analogues as fully ef-

Binding studies with rat pan-

plasma membranes

showed that binding was

rapid, reversible,

res-

urable, and high affinity. These studies indicate

“in-

the amino acid substitutions

introduced into these ana-

labeling. Because there is appar-

logues were well tolerated.

Although

a photolabile

region, permitting

importance

of

the

entire

native

secretinpeptide, we attempted to incorporate the residue for covalent attachment into distinct domains of the peptide. Our laboratory pNO,-Phe

Amylase release assays using dispersed rat acini confirmed

and

ligands that incorporate

trinsic” photoaffinity

secretin

chemically. pancreatic

secretin 27 were both

high-specific

idue into a pharmacophoric ent

identity

secretin receptor.28

as a photolabile

has successfully

used

residue that can be incorpo-

rated into a peptide probe for photoaffinity

labeling.13

In rat secretin, there is a single Phe residue in position 6, which is a logical place for a pNO,-Phe. In chicken secretin, there is an additional Phe in position 22, providing a second potential site to locate a pNO,-Phe. Based on the presence of a Tyr in position 10 in chicken secretin, we incorporated a similar residue into the synthetic probes as a site for their oxidative radioiodination. This would leave the important amino-terminal His free and underivatized. There was already precedent for incorporation of an iodo-tyrosine into the 10 position, although maintaining binding of such an analogue to the secretin receptor.38 Both peptides were characterized biologically and

temperature-dependent,

specific, satthat

their biological

activity and binding affinities are retained, the use of these probes in structural receptor characterization studies rests in their ability to covalently label the binding domain of the pancreatic secretin receptor. Indeed, the pN0,-Phe22 analogue specifically photoaffinity labeled a 50,000-62,000-molecular

weight protein

in

rat pancreatic plasma membranes. Covalent labeling of this protein was 50% inhibited by 4 nmol/L secretin and unaffected by up to 0.1 pmol/L VIP, confirming its high affinity and specificity for secretin. This rat pancreatic secretin receptor migrates on an SDSPAGE well below the position of VIP-preferring binding proteins labeled in rat pancreatic acini and the AR42J pancreatic adenocarcinoma cell line (mol wt, 60,000-80,000), and slightly below the 62,000-molecular weight secretin-binding protein labeled in rat gastric mucosa.‘2,39,40 These results essentially agree with the findings of Gossen et a1.,12 and have the added important advantages of specific “intrinsic” photoaf-

RECOMBINANT SECRETIN RECEPTOR

November 1993

Secretin peptidd, log M

VIP

1541

with a high affinity to rat pancreatic plasma membranes, it is unlikely that the introduction substitutions used substantially alters

‘0 -10 -9 -8 -7 -6‘‘0 -9 -8 -7

of any of the the secondary

structure of this peptide. It is more likely that Phe6 in the bound state is either oriented away from the binding domain

11692-

covalent

pNO,-Phe6.

p? 0

bound

67;

or faces it in such

intermolecular The

secretin

gand-binding tor is further

57-62 -m

issue

labeling

by light

of the orientation

27 will be better

200

activated of Phe6 in

addressed

domain of the pancreatic characterized. Recombinant Receptor I(

Figure 7. Photoaffinity labeling of membranes from transfected cells by the radioiodinated pN02-Phe” analogue. Membranes bearing the recombinant secretin receptor were incubated with radioligand in the presence of increasing concentrations of secretin and VIP and photolyzed for 30 minutes. The autoradiogram shows that covalent labeling of a 57,000-62,000-molecular weight protein was competed for in a concentration-dependent manner by secretin, with no reduction in this labeling in the presence of up to 1 pmol/L VIP. Two-nanomolar secretin inhibited 50% of specific labeling of this recombinant protein, similar to its IC, for inhibition of covalent labeling of the 50,00062,000-molecular weight protein in native pancreatic membranes. Labeling of BSA with M, = 67,000 was not competed for by either peptide. There was no saturable covalent labeling of any proteins in membranes from untransfected Cells.

a way as to prevent

as the li-

secretin

recep-

Pancreatic Receptor

-

116 -

70

67 -

T

X

r’ 45 -

finity

labeling

of the ligand-binding

ity to use plasma

membranes

acini, and more intense radiographic

exposure

as opposed

labeling times

domain,

allowing

42k

the abilto whole

shorter

auto-

(2-3 days compared

with

1 month). Our inability to covalently label this native rat pancreatic secretin-binding protein with pNO,-Phe6 analogue, despite membranes,

its high affinity raises

intriguing

the role of Phe6 in the binding tor. Residues

containing

binding

to rat pancreatic

possibilities of secretin

aromatic

and 6, His’ and Phe6, respectively,

concerning to its recep-

rings in positions

1

Endo F

+

-

-

+

-

-

Secretin

-

-

+

-

-

+

are felt to play a

major role in the full biological activity of this peptide.2’*24 Substitution of Phe6 by its D isomer, as well as reduction of the phenyl ring, markedly diminishes peptide affinity for secretin/VIP receptors in pancreatic, hepatic, and cardiac membrane preparations.21,4’,42 Some evidence suggests that Phe6 is important to the formation of a p sheet in the 6-8 region following the amino-terminal p turn.” As this analogue is both a fully efficacious secretagogue and binds

Figure 8. Enzymatic deglycosylation of affinity labeled native and recombinant secretin receptors. Membranes from native pancreas and recombinant receptor-bearing cells were photoaffinity labeled using radioiodinated pNO,-Phe2’ analogue and subsequently deglycosylated with endo F. Despite differences in the migration of the glycoproteins, the major protein core bands were of identical size, migrating at a molecular weight of 42,000. Aggregation of the deglycosylated proteins observed at the interface with the stacking gel resulted in reduced yields of core protein. Shown also are lanes representing the affinity labeling in the presence of competing 1 pmol/L secretin to show nonspecifically labeled bands that are not competed Off.

1542 ULRICHET AL.

The molecular receptors

GASTROENTEROLOGY

cloning and expression

has proven critical

receptor physiology

encoding

a secretin-binding

15)27 has provided new opportunities this recombinant

secretin

protein

beled, its relationship

for the investi-

receptor.

Although

had never been affinity la-

to the rat pancreatic

had yet to be determined.

secretin re-

In this work,

showed the cloning of a full-length

cDNA

we

from a rat

pancreatic library that was identical to that reported by Ishihara et a1.28 Successful expression was confirmed

clase in transfected

of this receptor

using both binding and photoaffinity

heart, stomach, portance

and central

of this receptor

In summary, that mimic

secretin

finity label the rat pancreatic an “intrinsic”

photolabile

in rat pancreatic

cloning of the cDNA encoding this receptor from a rat pancreatic

library, its expression

ex-

secretincovalent

of 2 nmol/L,

recombinant receptor

plasma

membranes

ther limited receptor

degradation

teases during membrane the posttranslational

covalently found

by pancreatic

eipro-

or variations

in

of this protein

in

of the secretin-binding

in both preparations,

The

likely represents

the two cell types. This identity was further by deglycosylation

repre-

membranes.

preparation

modification

shown proteins

to yield the same size protein

core. As noted earlier, open reading frame analysis of the cDNA

encoding

both native

forms of this receptor provide tools

to understanding

its structure

and function.

this protein predicted

ber of tissues,

knowledge

gained

using these tools

should lead to our better understanding

of the role of

this receptor

and nonpan-

in a variety of pancreatic

creatic disease states.

rat pancre-

almost certainly

slightly broader band (mol wt, 50,000-62,000) using rat pancreatic

in cultured cells, and

the ability of this analogue to characterize

in a num-

with an IC,,

secretin

secretin receptor through residue. Our confirmatory

binding protein identified and characterized

of this protein

sents the rat pancreatic

and

in membranes

labeling

labeled

activity

secretin-

whereas VIP had little effect. This 57,000-62,000-moprotein

in their biological

Because this may represent the high-affinity

this recombinant rat pancreatic protein. Cold secretin inhibited

lecular weight high-affinity

and

analogues

labeled a 57,000-

pressing binding

atic secretin-binding

the synthesis

binding affinities, with one able to efficiently photoaf-

and recombinant

only the pN02-Phe22

weight protein

nervous system, the im-

of two novel secretin-

critical

62,000-molecular

including

likely extends well beyond

we have reported

characterization

for both probes were similar,

photoaffinity

this G pro-

tein.28 With an apparent tissue distribution

labeling studies. Again, even though binding affinities analogue specifically

COS cells expressing

No. 5

the pancreas.

found in a hybrid neural cell line (NG108

gation of the pancreatic

ceptor

of

in a number of systems. The re-

cent cloning of a cDNA protein

of hormone

to our understanding

Vol. 105.

a 449-amino

acid (calculated

mol wt, 48,696)

receptor with seven

transmembrane

segments consistent with a G protein-

coupled receptor.28 Interesting features of this sequence include homology with the recently cloned calcitonin, parathyroid hormone, and VIP receptors 43,44 the absence of traditionally conserved residue: characteristic of the P-adrenergic family of G protein-coupled receptors, and a relatively large extracellular domain (121 residues) preceding the first transmembrane segment. The extracellular domain of this receptor contains multiple potential N-glycosylation sites, as well as a number of Cys residues, possibly important in ligand binding. Ishihara et al. showed that this protein likely interacts with G,, to achieve its high affinity state and that it activates adenylate cy-

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RECOMBINANT SECRETIN RECEPTOR

1993

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Received January 28, 1993. Accepted August 10, 1993. Address requests for reprints to: Laurence J. Miller, M.D., Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, Minnesota 55905. Supported by the Mayo Foundation as well as grants from the National Institutes of Health (DK32878 and DK07198). The authors thank Marilyn LeQve and Sara Erickson for their secretarial assistance.