THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 273, No. 17, Issue of April 24, pp. 10308-10312, 1998
`Printed in U.S.A.
`
`Positively Charged Residuesat Positions 12, 17, and 18 of
`Glucagon Ensure Maximum Biological Potency*
`
`(Received for publication, November 7, 1997, and in revised form, February 13, 1998)
`
`Cecilia G. Unson{, Cui-Rong Wu, Connie P. Cheung, and R.B. Merrifield
`From the Rockefeller University, New York, New York 10021
`
`Glucagon is a peptide hormonethat plays a central
`role in the maintenance of normal circulating glucose
`levels. Structure-activity studies have previously dem-
`onstrated the importanceof histidine at position 1 and
`the absolute requirement for aspartic acid at position 9
`for transduction of the hormonal signal. Site-directed
`mutagenesis of the receptor protein identified Asp™ on
`the extracellular N-terminal tail to be crucial for the
`recognition function of the receptor. In addition, anti-
`bodies generated against aspartic acid-rich epitopes
`from the extracellular region competed effectively with
`glucagon for receptor sites, which suggested that nega-
`tive charges may line the putative glucagon binding
`pocket in the receptor. These observations led to the
`idea that positively charged residues on the hormone
`may act as counterions to these sites. Based on these
`initial findings, we synthesized glucagon analogs in
`which basic residues at positions 12, 17, and 18 were
`replaced with neutral or acidic residues to examine the
`effect of altering the positive charge on those sites on
`binding and adenylyl cyclase activity.
`Theresults indicate that unlike N-terminal histidine,
`Lys!”, Arg!’, and Arg?® of glucagon havevery largeef-
`fects on receptor binding and transduction of the hor-
`monal signal, although they are not absolutely critical.
`They contribute strongly to the stabilization of the bind-
`ing interaction with the glucagon receptor that leads to
`maximum biological potency.
`
`Glucagonis a polypeptide hormonethatconsists of 29 amino
`acid residues and is a memberof a highly homologous family of
`biologically active peptides. Secreted by pancreatic A cells, its
`primary target organis the liver where, together with insulin,
`it plays a central role in the maintenance of normalcirculating
`glucose levels critical to the survival of the organism. The
`initial event in glucagonaction is bindingto its receptor on the
`surface of liver cells. The binding message constitutes the sig-
`nal that is transmitted across the membraneto guaninenucle-
`otide binding protein-linkedintracellular effectors that are ul-
`timately responsible for glucose production.
`The glucagon receptor is a memberof a unique branchof the
`G protein-coupled receptor superfamily that has highly homol-
`ogous sequences but shares very few of the conserved struc-
`tural features found within the rest of the G protein-coupled
`
`
`* This work wassupported by U. S. Public Health Service Grant DK
`24039. A preliminary report of part of this work was presented at the
`15th American Peptide Symposium, June 14-19, 1997, Nashville, TN.
`The costs of publication of this article were defrayed in part by the
`paymentof page charges.This article must therefore be hereby marked
`“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`+ To whom correspondence should be addressed: The Rockefeller Uni-
`versity, 1230 York Ave., New York, New York 10021. Tel.: 212-327-
`8239; Fax: 212-327-8245.
`
`receptor family (1, 2). Membersof this receptor group include
`receptors for the glucagon family of hormones, glucagon-like
`peptide 1 (GLP-1)(3), secretin (4), gastrin inhibitory peptide
`(5), and vasoactive intestinal peptide (6). The receptor has a
`relatively large extracellular N terminus thought to be in-
`volved in hormone-receptor interaction, followed by hydropho-
`bic helical segments postulated to span the membrane seven
`times and a cytoplasmic C-terminal domain (Fig. 1). Signal
`transduction is thought to proceed upon bindingof the hormone
`with the extracellular region of the receptor. The mechanism
`by which thesignal is conveyed from thecell surface across the
`transmembranehelical network to activate G protein-coupled
`effectors on the surface of cytoplasm is not understood.
`Extensive structure-function studies of glucagon have af-
`forded someinsight into the understandingof its mechanism of
`action. The general picture that has emergedis that the active
`pharmacophoreis dispersed throughout the glucagon molecule
`andthat the intact hormoneis necessary for the expression of
`full hormonal activity. Nevertheless, specific active site resi-
`dues responsible for either high affinity binding or activation
`have beensingled out. Electrostatic interactions of the nega-
`tively charged side chain of aspartic acid 9, 15, and 21 were
`shownto be essential in glucagon function (7, 8). Activity was
`lost when Asp? was deleted or replaced by any other amino
`acid.
`An important early finding from pioneering structure-activ-
`ity studies established that the N-terminal histidine which is
`strictly conserved within the glucagon peptide family was es-
`sential for receptor activation and less so for binding and im-
`plied that the deletion of histidine would produce a glucagon
`antagonist (9). Indeed, the first partial antagonists that were
`developed were des-His' derivatives or glucagon analogs that
`had modified histidines at the N terminus (10-11). Further
`studies demonstrated that the imidazole ring of histidine at
`position one of the hormone furnishes determinants for both
`receptor binding affinity and activity (12).
`Serine residues at positions 2, 8, and 16 were also shown to
`play prominent roles in glucagon action (13). The apparent
`connection of His', Asp®, and Ser?residues led to the hypoth-
`esis that a catalytic triad resembling that of a serine prote-
`ase might be involved in the mechanism of glucagon signal
`transduction (13).
`The glucagon binding cavity on the receptor is mostlikely a
`discontinuous domain that involves contributions from the long
`N-terminal extension as well as from the three extracellular
`loops that connect the seven transmembranehelices (14). In-
`formation about the complementary peptide andprotein inter-
`actions that dictate the binding phenomenonis central to the
`design of antagonists of the hormone that might beclinically
`relevant. To investigate the molecular mechanism of hormone-
`receptor interaction and of receptor activation by site-directed
`
`
`1 The abbreviation used is: GLP-1, glucagon-like peptide 1.
`
`10308
`
`This paperis available online at http://www.jbc.org
`
`This is an Open Accessarticle under the CC BYlicense.
`
`MSN Exhibit 1015 - Page 1 of 5
`MSNv. Bausch - IPR2023-00016
`
`

`

`Glucagon Structure and Function
`
`10309
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`Fic. 1. Schematic representation of
`the rat glucagon receptor primary
`Pp
`nq
`.
`and secondary structure. Seven puta-
`tive transmembranehelices based on pre-
`vious models of G protein-coupled recep-
`.
`tors are shown. The N terminus and
`extracellular surface is toward the top,
`1
`M
`and the C terminus and cytoplasmic sur-
`face is toward the bottom of the figure.
`Asp™, which was studied by site-specific
`mutagenesis,
`is numbered and labeled
`:
`2
`.
`with an arrow (15). The locations of se-
`quences from the extracellular domain
`that were used as the peptides for anti-
`receptor anti-peptide antibody production
`are boxed.
`
`mutagenesis, we have synthesized a genefor the rat glucagon
`receptor (15, 16).
`Theearliest information to come from site-directed mutagen-
`esis of the glucagon receptor protein identified Asp®™in the
`extracellular N-terminaltail to be absolutely required for the
`recognition function of the receptor (15). Recent studies have
`also implicated areas in the membraneproximalportion of the
`N terminus and thefirst extracellular loop to be part of the
`hormonebinding site (17). More importantly, antibodies raised
`against peptides representing sequences from these regions
`were inhibitors of glucagon bindingto the receptor (18). These
`peptide epitopes containedclusters of negatively charged resi-
`dues, which suggested that an electrostatic association with a
`complementary positively charged residue on the ligand might
`occur at these receptorsites.
`To test this possibility, we assessed the contribution of the
`positively charged groups at positions 12, 17, and 18, to gluca-
`gon receptor recognition, which to date has not been clearly
`established. We synthesized glucagon analogs containing sub-
`stitutions ofLys!?, Arg’, and Arg’® ofglucagon with neutralor
`negatively chargedresiduesto examinetheroles ofthe positive
`charge on those sites on binding and adenylyl cyclase activity.
`It was reported in an earlier study of the C-terminal region of
`the hormone that the analog [Lys'’,Lys'®,Glu?"]glucagon ex-
`hibited enhancedreceptor binding and was a superagonist(19).
`This behavior was attributed to an increased a-helical content
`and the possible formation of an intramolecular salt bridge
`between charged side chains at positions 18 and 21 (20, 21).
`Our results demonstrate that glucagon binding andactivity are
`not dictated solely by electrostatic interactions but include the
`interactions of hydrophobic side chains with the receptor.
`EXPERIMENTAL PROCEDURES
`
`Peptide Synthesis and Purification—Thirty three analogs of glucagon
`with replacements at positions 12, 17, and 18 were assembled by the
`solid-phase method (22, 23), on an Applied Biosystems 430A peptide
`synthesizer, using procedures previously described for the synthesis of
`glucagon analogs (24). Briefly, the peptide analogs with C-terminal
`amides were prepared on p-methylbenzhydrylamine-resin (Peptides In-
`ternational, 0.66 mmol/g) using N*-Boc [tert-butyloxycarbonyl] protec-
`tion chemistry. N%-Boc-protected amino acids were purchased from
`Peptide Institute. Side chain protection was Arg(Tos), Asp(OcHx), Glu-
`(OcHx), His(Tos), Lys[Z(CD], Ser(Bzl), Thr(Bzl), Trp(For), and Tyr-
`[Z(Br)] (where Tosis tosyl; cHx is cyclohexyl; Z(Cl) is 2-chlorobenzyl-
`oxycarbonyl; Bzl is benzyl; and For is formyl). Standard protocol for
`double couplings with preformed symmetric anhydrides in dimethylfor-
`mamide were used routinely, except for arginine, asparagine, and glu-
`
`tamine which were coupled as N1-hydroxybenzotriazole esters (25). The
`N*-formyl group on tryptophan was removed with 50% piperidine in
`dimethylformamide, prior to HF treatment. After cleavage by anhy-
`drous HF, the crude peptides were purified by preparative low pressure
`reverse-phase liquid chromatography on octadecyl-silica (Vydac C18,
`Separations Group). The peptides were eluted by applying a linear
`gradient of 25-40% acetonitrile in 0.05% trifluoroacetic acid. Purity of
`the lyophilized product was evaluated by analytical high pressure liq-
`uid chromatography (Vydac 218TP54) in at least two different solvent
`systems and massspectral analysis by the electrospray methodidenti-
`fied the expected (M + H)* peaks within +0.3 Da. Aminoacid analysis
`yielded amino acid compositions consistent with theory.
`Receptor Binding Assay—Liver plasma membranes were prepared
`from rat liver (Sprague-Dawley, 100-150 g, Charles River) following
`the method of Neville with modifications described by Pohl (26). Mem-
`brane aliquots were stored in liquid nitrogen and used within 4-6
`months. The receptor binding assay was done according to Wright and
`Rodbell (27), in which competition for glucagon receptors in 10 pg of
`liver membraneprotein, between '”°I-labeled glucagon (NEN Life Sci-
`ence Products) (1.6 nM) and the synthetic analogs in concentrations
`ranging from 10711 to 107° M, was measured. Assay suspensions were
`filtered on Durapore membranefilters (0.45 wm) using a vacuum fil-
`tration manifold (Millipore). Binding affinity (percent) is calculated as
`the ratio of the concentration of glucagon that inhibits 50% of the
`binding of tracer (IC5,) to that of peptide analog x 100. Duplicate
`determinations were made for each concentration point, and each assay
`wasrun at least twice. Nonspecific binding, determinedin the presence
`of 10° M unlabeled glucagon, wastypically 10% of total binding.
`Adenylyl Cyclase Assay—Adenylyl cyclase activity was measured
`according to the procedure described by Salomonet al. (28). cAMP
`released was determined with a commercially available assay kit, from
`Amersham Pharmacia Biotech, in which unlabeled cAMP produced
`competes with [8-H]cAMP for a cAMP-binding protein. Data for stim-
`ulation of adenylyl cyclase are expressed as picomoles of cAMP pro-
`duced per mg of membrane protein per min and plotted against the
`logarithm of peptide analog concentration. Maximum activity (percent)
`of an analog is the percentage of maximum stimulation of cAMP pro-
`duction abovebasal by glucagon. Relative potency (percent) is the ratio
`of the concentration of natural glucagon that elicits 50% maximum
`production of cAMP (EC,,) to that concentration of peptide analog x
`100. Inhibition of CAMP production was determined in a similar assay
`in which a constant amount of glucagon is allowed to compete with
`increasing concentrations of analog. The inhibition index (I/A);, is de-
`fined as the ratio of the concentrationsof inhibitor to agonist when the
`response is reduced to 50% of the response to agonist in the absence of
`inhibitor. Analogs were tested for inhibitory properties if they had
`relative potencies of =1% and a binding affinity of =1%. The pA, value,
`calculated by the method of Arunlakshana and Schild (29), is the
`negative logarithm of the concentration of inhibitor that reduces the
`response to 1 unit of agonist to the response produced by 0.5 unit of
`agonist. Duplicate determinations were made for each concentration
`point, and each experiment wascarried outat least twice.
`
`MSN Exhibit 1015 - Page 2 of 5
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`

`10310
`
`Glucagon Structure and Function
`TABLE I
`Glucagon analogs with neutral residue replacements at positions 12, 17, and 18
`Adenylyl cyclase activity
`
`
`
`
`
`Bindin,
`Analog of glucagon amide®
`
` ofa od Relate Wags pad
`
`%
`%
`%.
`15
`100
`100
`Glucagon amide
`15.8
`17.3 + 0.2
`59.7
`1. Ala?”
`0.15
`0.91
`12.5
`2. des-His',Ala’™”
`13.8
`11.4
`85.1
`3. Gly?
`0.13
`0.58
`19.4
`4, des-His',Gly'”
`31.6
`47 +1
`90.5
`5. N*-acetyl-Lys’”
`0.013
`38
`29
`6. Ala?”
`0.28
`2.3
`28
`7. des-His*,Ala’”
`37.1 + 1.7
`30+1.8
`88+ 2
`8. Leu?”
`2.13
`9.3
`23
`9. des-His',Leu'”
`70.8
`13
`94.4
`10. Ala'®
`0.14
`3.1
`14
`11. des-His*,Ala?®
`45.7+ 1.6
`56 + 1.5
`95 +3
`12. Leu’®
`1.5
`3.6
`22.5
`13. des-His?,Leu?®
`27.5
`8
`97
`14, Alat”,Ala?®
`0.19
`0.32
`10
`15. des-His',Ala’’,Alal®
`52.5 + 1.2
`7+0.1
`85.4 + 0.8
`16. Leu?’,Leu'®
`7.1
`43.7
`1.15
`1
`17
`17. des-His',Leu!’,Leu'®
`
`18. Ala!?,Ala’”,Ala!® 13 0.08 62.8
`
`
`* Analogs of glucagon amide were assayed using native glucagon as the standard, in both membrane binding and adenylyl cyclase activity.
`[des-His']Glucagon amide hada bindingaffinity of 63% and a relative potency of 0.16% in the adenylyl cyclase assay.
`> Bindingaffinity (%) is the ratio of agonist concentration to analog concentration at 50% receptor occupancy (IC5,) x 100.
`©“ Maximumactivity (%) is the percentage of maximum glucagon stimulation of cAMP production above basal.
`@ Relative potency (%) is the ratio of glucagon concentration to analog concentration at 50% response (EC,,) x 100.
`© The inhibition index (I/A);, is the ratio of peptide inhibitor concentration to glucagon concentration whenthe responseis reduced to 50% of the
`response of agonist in the absence of inhibitor.
`f The pA, valueis the negative logarithm of the concentration of inhibitor that reduces the responseto 1 unit of agonist to the response obtained
`from 0.5 unit of agonist.
`
`43.7
`
`34.7
`
`43.7
`
`85.1
`
`7.0
`
`7.5
`
`7.3
`
`7.0
`
`RESULTS
`
`Thirty three glucagon analogs have been synthesized to as-
`sess the roles of the positively charged basic residues at posi-
`tions 12, 17, and 18 of glucagon, in receptor binding affinity as
`well as in adenylyl cyclase activation. Ourinitial approach was
`to examine the effect of neutralizing the positive charge by
`substituting uncharged aminoacidsat positions 12, 17, and 18
`(Table I). Replacing Lys’? with neutral residues in the analogs
`Ala! and Gly?” glucagon amides (analogs 1 and3) resulted in
`an 80-90% reduction in bindingaffinity relative to glucagon for
`the glucagon receptor in rat liver membranes. However, both
`analogs werestill capable of a full agonist response, with re-
`duced potency. Similarly, acetylation of the «amino group of
`Lys” provided [N‘-acetyl-Lys?]glucagon amide (analog 5),
`which bound with 47% affinity and elicited 90% maximum
`adenylyl cyclase stimulation. These results were consistent
`with an earlier observation that N‘-acylated derivatives of
`glucagon werefull agonists (30) and further implicated a pref-
`erence for a hydrophobic functional group at position 12. An
`exchange of Arg?” for alanine or leucine in analogs 6 and 8
`(Table I) effected a loss of 60-70% bindingaffinity. Ala!’ was a
`weak agonist, whereas Leu?” stimulated adenylyl cyclase with
`an 88% maximum activity. Replacing Arg’® with alanine (an-
`alog 10) led to an 87% loss in binding, whereas a more hydro-
`phobic leucine substitution (analog 12) suffered a smaller loss
`of 44% affinity. Both analogs were capable of a full agonist
`response. Substitution of both sequential arginines with a neutral
`aminoacid in Ala‘”,Ala’® (analog 14) and Leu?’,Leu’® (analog 16)
`resulted in a 90% loss of binding, which in the case of Leu?’,Leu’®
`appeared to be additive. The concurrent loss of both positive
`charges did not influence the ability to activate adenylyl cyclase
`since the doubly substituted analogs were full agonists but with
`lowered potency. In contrast, the exchangeof all three basic resi-
`dues with alanines in [Ala!’,Ala’’,Ala']glucagon amide induced
`almost complete loss ofbinding and resulted in a very weak partial
`agonist. Deletion of Hist from someof the analogs that retained
`
`good bindingaffinities (analogs 6, 8, 10, 12, and 16), produced the
`corresponding des-His? derivatives (analogs 7, 9, 11, 13, and 17)
`that exhibited lowered potencies and measurable antagonist prop-
`erties, which is consistent with the establishedrole of histidine in
`glucagon (9-12). Because these des-His? analogs retained the ca-
`pacity to induce low levels ofcAMP,they were only partial glucagon
`antagonists (31).
`Aside from neutral amino acids, positive residues at posi-
`tions 12, 17, and 18 were each replaced with an aspartic or
`glutamic acid to examine theeffect of a reversal of charge.
`Asp’? and Glu’? (analogs 19 and 20, Table II) displayed poor
`bindingaffinities of 0.6 and 1%, respectively. Likewise, substi-
`tution by aspartic acid at positions 17 or 18 as in [Asp?”]- and
`[Asp?®]glucagon amides (analogs 22 and 26)led to 99% loss of
`binding. Interestingly, glutamic acid was better tolerated at
`these positions, with Glu!” and Glu’® glucagon amides (analogs
`24 and 28) exhibiting a retention of 21 and 6% bindingaffinity,
`respectively, and full stimulation of adenylyl cyclase. Despite
`reduced bindingaffinities a reversal of charge at all positions
`produced glucagon analogs that elicited substantial agonist
`responsesalthough with reduced potencies. Unlike Alat’,Ala?®,
`however, a double replacement with acidic residues in
`[Asp!”,Asp?°]- and in [Glu?”,Glu?®]glucagon amides(analogs 30
`and 32) rendered the peptides incapable of receptor recogni-
`tion. Since it is acknowledged that an intact N-terminalhisti-
`dine provides determinants for both the binding andactivation
`function of glucagon, the des-histidine derivative of every po-
`sition —12, —17, and —18 replacement analog predictably lost
`additional receptor binding affinity and potency of adenylyl
`cyclase activation.
`
`DISCUSSION
`
`There is renewed interest in the peptide glucagon because of
`its role in diabetes mellitus. Despite considerable positive evi-
`dence, the participation of glucagon is still somewhat contro-
`versial and further evidencefor its role is needed. It has been
`
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`10311
`
`57.5
`
`6.4
`
`Glucagon Structure and Function
`TABLE II
`
`Glucagon analogs with acidic residue replacements at positions 12, 17, and 18
`
`.
`Adenylyl cyclase activity
`Analog ofglucagon amide®
`Binding
`Maximum
`Relative
`WA)ac?
`Af
`
`activity®
`poten
`50
`PAs
`%
`%
`%
`100
`100
`15
`Glucagon amide
`0.6
`78.4
`10
`19. Asp?
`1
`80.4
`50.1
`20. Glu’?
`0.11
`28
`0.22
`21. des-His',Glu”
`1.4
`82.4
`44
`22. Asp?”
`0.1
`11.5
`0.08
`23. des-His',Asp*”
`21.3 + 0.5
`94.8 + 0.2
`40.7+3
`24. Glu!’
`1.7
`21.5
`1.0
`25. des-His',Glu’”
`0.22
`69.2
`0.24
`26. Asp?®
`<0.038
`27. des-His',Asp1®
`6.2 + 0.2
`28. Glu!
`0.44
`29. des-His',Glu’®
`<0.032
`30. Asp?’,Asp?®
`<0.032
`31. des-His*,Asp?”,Asp?®
`1.2
`100
`0.036
`32. Glu?”,Glu’®
`33. des-His’,Glu’’,Glu'® <0.050
`
`* Analogs of glucagon amide were assayed using native glucagon as the standard, in both membrane binding and adenylyl cyclase activity.
`[des-His']Glucagon amide hada binding affinity of 63% and a relative potency of 0.16% in the adenylyl cyclase assay.
`> Bindingaffinity (%) is the ratio of agonist concentration to analog concentration at 50% receptor occupancy (IC;,) x 100.
`° Maximum activity (%) is the percentage of maximum glucagon stimulation of cAMP production above basal.
`4 Relative potency (%) is the ratio of glucagon concentration to analog concentration at 50% response (EC,,) < 100.
`© The inhibition index (I/A);, is the ratio of peptide inhibitor concentration to glucagon concentration when the responseis reduced to 50% of the
`response of agonist in the absenceof inhibitor.
`f The pA,valueis the negative logarithm of the concentration of inhibitor that reduces the responseto 1 unit of agonist to the response obtained
`from 0.5 unit of agonist.
`
`100 + 2
`18
`
`3.3 + 0.3
`0.24
`
`observed that overproduction of glucose by elevated circulating
`levels of glucagon may be a contributing factor to hyperglyce-
`mia and ketoacidosis that is characteristic of the disease (32,
`33). It was reasonable to assume that antagonists of the hor-
`mone that are able to inhibit the actions of this endogenous
`glucagon by competing for the same binding cavity in the
`glucagon receptor could haveclinical potential in the manage-
`ment of diabetic complications (9, 32, 33). Indeed, several pep-
`tide analogs have been developed that have been shown to
`effectively inhibit the effects of glucagon both in vitro and in
`vivo (24, 31, 34-38). Continuedefforts in the study of glucagon
`are spurred by the idea that the ability to single out specific
`contact points between the peptide ligand and its receptor
`protein would serve as a basisfor the rational design of analogs
`that bind yet do not activate adenylyl cyclase.
`This study of the electrostatic interaction of the basic resi-
`dues Lys”, Arg”, and Arg?® of glucagon with acidic residues of
`the glucagon receptor is based on the supposition that any one
`or all of these groups may provide a counterion to a specific
`aspartic acid residue on the extracellular domainof the recep-
`tor that has been shown in recent mutagenesis studies to be
`critical for ligand recognition (15, 18). The importanceofhisti-
`dine at position 1 to both receptor binding andactivation has
`been firmly established (9-12). Removal of the histidine group
`afforded an analog that retained affinity for the receptor but,
`more importantly, appeared to partially inhibit glucagon-stim-
`ulated adenylyl cyclase (9). Although the N-terminalhistidine
`is strictly conserved within the glucagon family of peptide
`hormones, Lys!”, Arg!’, and Arg!® are relatively unique at
`these positions in glucagon and mightalso serve as determi-
`nants of receptor specificity.
`The results of our study reveal that while neutral residue
`scanning of positions 12, 17, or 18 of glucagon strongly atten-
`uated receptor binding, most of the resulting analogs were
`weak but full agonists, suggesting that a positive charge at
`these particular positions was not absolutely critical for activ-
`ity. That no positively charged aminoacids of glucagon, with
`the exception of histidine 1, are critical for the activation func-
`tion is indicated by the observation that none of the replace-
`
`glucagon |H S|Q|G TFT SD/Y/S KIY LIDJS R AJAQ DIF VQIWIL MIN T -
`
`
`
`|H[AJEG TF TSDIV S[SY L GQ AJA|K E/F I[AWILV|IKGR
`gip!
`
`secretin TSO GET SEL SAG SARILQ RIL LQIG/L VI- - -
`vip [HS DJA VLE TIDN Y TIRJLR KQM ALVJK KLY_ LNJSLI LIN - -
`
`Consensus HSDGTFTSD-SR--D---A--FLQ-LV---
`
`
`
`Fic. 2. Sequence alignmentof peptides of the glucagon family
`that have close homology. Residues that are conserved in either
`charge or hydrophobic character are boxed.
`
`ment analogs in Table I behaved as antagonists. However,
`someof the corresponding des-His! derivatives of positions 17
`and 18 replacement analogs displayed partial antagonist prop-
`erties, which was therefore associated with the deletion of
`position 1 histidine (31). These were in the (I/A);, range of
`34-85, whereas the most potent glucagon antagonist reported
`to date was 0.85 (36).
`A negatively charged acidic amino acid was, however, less
`tolerated at these positions and impaired receptor binding by a
`hundred-fold or more. Strongly reduced bindingaffinities were
`coupled with adenylyl cyclase responses with much weakened
`potencies. Thus, a positive charge at these positions is neces-
`sary for optimal hormonal function.
`Our previous findings established the roles of the aspartic
`acid residues at positions 9, 15, and 21 of glucagon. Asp? is
`critical for transduction but not for binding (7), whereas the
`negative charge at Asp?®is absolutely essential for binding(8).
`In contrast, the positively charged residues in the central re-
`gion of the hormonehave a specific role in achieving optimal, or
`even significant, binding and maximalbiological potency. An
`alteration in the charge distribution along the molecule clearly
`results in decreased binding of the derivatives. Unlike posi-
`tions 9 and 15 wheretheprecise location of an aspartic acid
`residue is critical to hormone-receptor interaction, it appears
`that glucagon binding affinity is not regulated by the topo-
`graphic location of a specific positive charge but by a net pos-
`itive charge. An overall positively charged molecule definitely
`enhancestheaffinity for its membrane-boundreceptorprotein.
`A single replacement in glucagon amide with an uncharged
`residue did not adequately alter the overall charge to reduce
`MSNExhibit 1015 - Page 4 of 5
`MSNv. Bausch - IPR2023-00016
`
`

`

`10312
`
`Glucagon Structure and Function
`
`binding andactivity, but one substitution with a negatively
`charged residue resulted in a neutral molecule.
`Secretin, GLP-1, and vasoactive intestinal peptide, gluca-
`gon’s closest relatives within the family of peptide hormones,
`share 50% sequence homology mostly at the N-terminal half of
`the molecule (Fig. 2). With the exception of His", the positions
`of basic residues scattered primarily along the C-terminal part
`of these sequences are not well conserved. In an earlier report,
`a glucagon-GLP-1 chimeric peptide, in which thefirst 14 resi-
`dues of glucagon were combined with the last 16 residues of
`GLP-1, bound to both glucagon and GLP-1 receptors (39). The
`normal peptides only bind weakly to each other’s receptor.
`Interestingly, what appears to be preserved is an overall posi-
`tive charge, suggesting that a positively charged ligand may be
`one requisite feature common to membersof this G protein-
`coupled receptor sub-group andtherefore not a strict determi-
`nant of specificity.
`The study also reveals a hydrophobicity component of the
`bindinginteraction. The basic amino acids arginine andlysine
`can contribute not only a charged group but also an aliphatic
`component to the polar and non-polar interface of the ligand
`binding pocket. The proposed ligand binding site should lie
`within a hydrophobic core of the receptor where nonspecific
`hydrophobic interactions between the hormoneandits receptor
`embedded in the membranebilayer augment bindingaffinity.
`This explains whysubstitution with a neutral yet hydrophobic
`molecule like alanine or leucine was well tolerated despite the
`loss of a positive charge and could sustain 30-60% of the
`affinity for the receptor. A reversal of charge on the other hand
`adversely altered the polar character of the peptide and led to
`a greater loss of receptor recognition (Table II). Nevertheless,
`an increased hydrophobic contribution from a glutamic acid
`side chain probably compensatedfor the reversal of charge and
`accounted for the retention of substantial binding affinity and
`potency of the Glu?” and Glu’® replacement analogs (analogs
`24 and 28, Table II). In addition, des-His',Glu’” (analog 25,
`Table II), which retained 2% binding and a weakened potency
`due to deletion of histidine, wasstill a partial antagonist. The
`analog [Lys?’,Lys?®,Glu”"]glucagon has been reported to be a
`superagonist which bound 5-fold better than the natural hor-
`mone and had a higherpotency(19, 20). A recent x-ray crystal
`structure for [Lys!’,Lys?®,Glu”|glucagon suggested that the
`formation of a salt bridge between the e-amino group of Lys'®
`and the carboxyl of Glu?! maystabilize the turn of a putative
`a-helix at residues 18—21 and contributes to its superagonist
`activity (21). However, bindingaffinity remains relatively high
`even when hydrophobic residues are substituted for either
`Arg?” or Arg!®. Presumably, enhanced activity may also be
`attributed to the increased hydrophobicity of the longer ali-
`phatic side chains of lysine and glutamic acid relative to those
`of the normal arginine and aspartic acid residues.
`Thus, the positively charged amino acids Lys??, Arg!”, and
`Arg?® of glucagon have large effects but are not absolutely
`critical for the binding and activation function of the hormone.
`However,the functional groups of these basic residues bolster
`both the polar and non-polar aspects of the peptide and protein
`interactions that occur within the receptor binding site and
`ensure maximumbiological activity. The aliphatic backbone of
`arginine andlysine residues optimize ligand “fitting” within a
`hydrophobic pocket in the receptor. Our data demonstrate that
`at these positions in glucagon, nonspecific hydrophobic inter-
`
`actions are as importanta contributing factor to bindingaffin-
`ity as the electrostatic effects. Mutagenesis studies on the
`receptor have outlined the perimeter of a putative bindingsite
`bordered by negatively charged residues. Thus, an overall pos-
`itively charged glucagon molecule contributes to the stabiliza-
`tion of the hormone-receptor complex and secures the binding
`conformation that subsequently leads to activation.
`
`REFERENCES
`1. Jelinek, L. J., Lok, S., Rosenberg, G. B., Smith, R. A., Grant, F. J., Biggs, S.,
`Bensch,P. A., Kuifper, J. L., Sheppard, P. O., Sprecher, C. A., O’Hara,P. J.,
`Foster, D., Walker, K. M., Chen, L. H. J., Mckernan, P. A. & Kindsvogel, W.
`(1993) Science 259, 1614-1616
`2. Segre, G. V. & Goldring, S. R. (1993) Trends Endocrinol. Metab. 4, 309-314
`3. Thorens, B. M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8641-8646
`4. Ishihara, T., Nakamura, S., Kaziro, Y., Takahashi, T. Takahashi, K. & Nagata,
`S. (1991) EMBOJ. 10, 1635-1641
`5. Usdin, T. B., Mesey, E., Button, D. C., Brownstein, M. J. & Bonner, T. J. (1993)
`Endocrinology 133, 2861-2870
`6. Ishihara, T., Shigemoto, R., Mori, K. & Nagata, S. (1992) Neuron 8, 811-819
`7. Unson, C. G., Macdonald, D., Ray, K., Durrah, T. L. & Merrifield, R. B. (1991)
`J. Biol. Chem. 266, 2763-2766
`8. Unson, C. G., Wu, C.-R. & Merrifield, R. B. (1994) Biochemistry 33, 6884-6887
`9. Lin, M. C., Wright, D. E., Hruby, V. J. & Rodbell, M. R. (1975) Biochemistry 14,
`1559-1563
`10. Hruby, V. J. (1982) Mol. Cell. Biochem. 44, 49-64
`11. Hruby, V. J., Krstenansky,J. L., McKee, R. & Pelton, J. T. (1986) in Hormonal
`Control of Gluconeogenesis (Kraus-Friedman, N., ed) Vol. 2, pp. 3-20, CRC
`Press, Inc., Boca Raton, FL
`12. Unson, C. G., Macdonald, D. & Merrifield, R. B. (1993) Arch. Biochem. Bio-
`phys. 300, 747-750
`13. Unson, C. G. & Merrifield, R. B. (1994) Proc. Natl. Acad. Sci. U. S.A. 91,
`454-458
`14. Unson, C. G., Cypess, A. M., Kim,