Pharmacological Characterization and Investigation of N-Terminal

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Pharmacological characterization and investigation of N-terminal loop amino acids of adrenomedullin 2 that are important for receptor activation Hala Musa, Erica Hendrikse, Margaret A. Brimble, Michael Garelja, Harriet A Watkins, Paul W. R. Harris, and Debbie L. Hay Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00571 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Biochemistry

Pharmacological

characterization

and

investigation of N-terminal loop amino acids of adrenomedullin 2 that are important for receptor activation. Hala Musa1, Erica R. Hendrikse1, Margaret A. Brimble1,2,3, Michael L. Garelja1, Harriet A. Watkins1, *Paul W.R. Harris1,2,3, *Debbie L. Hay1,2 1. School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland, 1010, New Zealand. 2. Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3A Symonds Street, Auckland, 1010, New Zealand. 3. School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland, 1010, New Zealand.

KEYWORDS. G protein-coupled receptor, Receptor activity-modifying proteins, calcitonin receptor-like receptor, adrenomedullin, calcitonin gene-related peptide, adrenomedullin 2, intermedin. ABSTRACT: Adrenomedullin 2 (AM2) is a peptide hormone with potent effects in the cardiovascular system. The N-terminal disulfide loop of AM2 is thought to be important for interacting with its receptors to initiate a signaling response. However, the relative contribution of each amino acid within this region is currently unknown. Thus, the region was investigated using an alanine scanning approach. Two AM2 peptides (AM2-47 and AM2-40) were directly compared at the CGRP, AM1 and AM2 receptors in transfected Cos7 cells and found to have equivalent activity. Analogs of AM2-40 were then synthesized, substituting each individual amino acid within the disulfide loop with alanine. The ability of these analogs to stimulate a cAMP response was evaluated at the CGRP, AM1 and AM2 receptors. AM2-40 L12A and T14A had reduced abilities to elicit cAMP responses through all tested receptors. In contrast, AM2-40 G13A was slightly more potent than the unmodified peptide at all tested receptors. Thus, it appears that residues within the disulfide loop region play differential roles in the ability of AM2 to stimulate cAMP production. The data provide the first structure-function investigation of AM2 agonism.

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Introduction Adrenomedullin 2 (AM2)/intermedin was independently discovered by two groups in 20041, 2. Given that intermedin was the former name for α-melanocyte stimulating hormone, AM2 is the preferred nomenclature3. The amino acid sequences of AM2 peptides are shown in Figure 1, being produced from a pre-pro hormone3. There are three resulting peptides of 40, 47, and 53 amino acids in length, named as AM2-40, AM2-47 and AM2-53 respectively3. The additional amino acids are found as extensions at the N-terminus of AM2 (Figure 1). AM2 is most closely related to adrenomedullin (AM) and belongs to the broader calcitonin (CT) gene-related peptide (CGRP)/CT peptide family. AM2 is found in peripheral tissues and in the central nervous system. AM2 is found in the brain (especially the hypothalamus), pituitary, heart, lung, pancreas, spleen, thymus, kidney, stomach, ovary and circulating in plasma4. It has several effects in the cardiovascular system. For example, peripheral administration decreases aortic resistance, improves heart performance and elevates coronary perfusion flow under physiological conditions 5. There are several reports of protective effects, with AM2 being reported to reduce the size of a myocardial infarction6, 7. Centrally, AM2 has effects on regulating prolactin, oxytocin, and vasopressin8, 9. As such, there is interest in ascertaining which receptors mediate its actions, and in understanding its structure-function relationships to aid drug development.

Figure 1. Amino acid sequence alignment of AM2 peptides and its alanine scan analogs. The underlined C-terminal tyrosine is an amide. A disulfide bond is formed between the two bolded cysteines. Residues substituted with alanine in this study have been shaded gray. The numbering on the bottom line refers to the residue number within AM2-47. As a member of the CGRP/CT-family of peptides, it is expected to share receptors with these peptides. Two G protein-coupled receptors (GPCRs), the CT receptor (CTR) and the calcitonin receptor-like receptor (CLR) form dimers with three receptor activity-modifying proteins (RAMPs) to create receptors that are activated by this peptide family4. AM2 has weak activity at human CTR based receptors, though it is worth noting its activity at the rat CTR/RAMP3 complex10, 11. CLR with RAMP1 gives the CGRP receptor and with RAMP2 or 3, produces the AM1 or AM2 receptors, respectively. Currently, the nomenclature of the AM2 receptor does not relate to the specific ability of the peptide AM2 to activate this receptor, although this does appear to be the receptor at which AM2 is most active, when measuring cAMP production4. Within this receptor family, peptides bind to both the extracellular domain (ECD) and the transmembrane domain. The transmembrane domain is generally thought of as the component most responsible for triggering receptor activation. A recent cryo-electron microscopy structure of CGRP bound to CLR/RAMP1 has given detailed insights into how this particular ligand-receptor combination functions12. However, for AM2 in particular there is very little information regarding ligand binding and receptor activation mechanisms. A recent report


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Biochemistry

revealed details of the AM2 C-terminus binding to the ECD of the CLR/RAMP1 complex, which showed interesting differences in the way that CGRP, AM and AM2 bind to CLR/RAMP ECD complexes13. Additionally, chimeras of AM, CGRP and AM2 have been shown to offer altered selectivity at CLR/RAMP complexes relative to their unmodified counterparts indicating that the different peptides interact with receptors with unique conformations14. Studies investigating receptor mutants have shown that different regions on the receptor are important for different peptides, again highlighting how each peptide is likely to interact with the receptor in different conformations15, 16. Thus, while it is anticipated that the AM2 N-terminus will play a critical role in receptor activation, there is currently no molecular information to support this. Therefore the aim of this study was to probe the mechanism by which AM2 stimulates receptor activation by performing an alanine scan of its N-terminus. AM2 can theoretically exist as one of three peptide lengths, being 40, 47, or 53 amino acids in length3. These peptides have similar effects in vivo, though there are reports of relative effectiveness differing across tissue types1, 17, 18. High-performance liquid chromatography has isolated AM2-47 in human tissues, however other peptide lengths have yet to be identified in vivo18, 19. In this study, the pharmacology of AM2-47 was compared to that of AM2-40. AM2-47 was chosen for investigation as this is the longest peptide fragment identified in vivo. AM2-40 was chosen for comparison as this length is the most similar in length to other CGRP/CT family peptides4, and there are numerous examples of N-terminally truncated derivatives of AM which have comparable pharmacology to AM (for example, AM13-52, and AM15-52)2022.

Following this, individual amino acids within the AM2-40 disulfide loop were sequentially replaced with alanine (Figure 1). Residues

chosen for investigation were within the N-terminal disulfide loop, as work utilizing truncated peptides has shown that the disulfide loop region is critical for the activity of AM2 and the entire peptide family13. The ability of these analogs to stimulate cAMP production was investigated at all three CLR based receptors. This research gives us insight into residues within the AM2 N-terminus which are important for its activity.

Methods Peptides - Human AM2-47 was purchased from Bachem (Bubendorf, Switzerland). Human AM and human αCGRP were purchased from American Peptide (Sunnyvale, CA, U.S.A.). AM2-40 and analogs were synthesized in-house using Fmoc solid-phase approaches (for further information on peptide synthesis, see Supporting Information). Peptides synthesized in-house were assumed to have a peptide content of 80%. All peptides were reconstituted in H2O and stored in protein LoBind tubes (Eppendorf, Hamburg, Germany) at -30˚C. Peptides experienced a maximum of two freeze-thaw cycles.

Expression constructs - Human HA-tagged CLR (Uniprot Q16602) was used throughout this study as it has been shown to be comparable to untagged receptor23. Human RAMP1, RAMP2, and RAMP3 used in this study were untagged (Uniprot O60894, O60894 and O60896, respectively).

Cell Culture - Cos7 cells were used in this study as they lack endogenous expression of CLR, CTR, and RAMPs23. Cell culture was performed as previously described 23. Briefly, cells were grown in DMEM supplemented with 8% heat-inactivated fetal bovine serum and grown in a 37°C, 5% CO2 humidified incubator. Cells were seeded at 20,000 cells per well in 96 well Spectraplates (PerkinElmer). Cells


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were transfected the day after plating using polyethylenimine and a 1:1 ratio of CLR:RAMP DNA. Cells were left to grow in transfection mix for two days before being used in signaling experiments.

cAMP Assays - Cells were stimulated with agonists in triplicate as previously described with minor modifications24. Briefly, 36-48 hours after transfection, cells were serum starved at 37˚ C for 30 minutes in cAMP assay media (DMEM + 0.1% bovine serum albumin + 1 mM 3-isobutyl-1-methylxanthine). Cells were then stimulated with a range of peptide concentrations (diluted in cAMP assay media) for 15 minutes. Media was then aspirated from the wells and ice-cold absolute ethanol added. cAMP production was measured using a LANCE assay, with the second incubation step reduced to one hour (Perkin-Elmer).

Data analysis - Analyses were performed in GraphPad Prism. cAMP values were interpolated from a standard curve that was included in each experiment. Concentration-response curves were fitted with a 3-parameter logistic equation, from which pEC50 values were derived. Potency differences between CGRP, AM, AM2-40 and AM2-47 were analyzed using a one-way ANOVA with post-hoc Dunnett’s test, comparing the potency of each peptide to AM2-40. Potency differences between AM2-40 and analogs were analyzed using unpaired t-tests because all experiments included a control AM2-40. This created an experimental design where results for individual analogs were linked to the results of the unmodified AM2-40 included on each plate, and not to the other analogs. Normalized Emax values were obtained by normalizing data from each experiment to the Emax and Emin of AM2-40 in each experiment, 95% confidence intervals were used to determine significance as previously described15, 24. Normalized data sets were generated by combining the mean of data points from individual experiments.

Results and Discussion AM2-40 and AM2-47 are functionally equivalent at CLR based receptors in Cos7 cells. The ability of AM2-40 and AM2-47 to stimulate cAMP production through CLR based receptors was compared to investigate whether there were any functional differences arising from the N-terminal extension of the peptide. These peptides were also compared to other peptides from the family, namely AM and CGRP. The relative rank order of potency at the CGRP receptor was CGRP>AM2-40=AM247>AM. There were no significant differences in Emax at this receptor (Figure 2, Table 1). At the AM1 receptor the relative rank order of potency was AM>AM2-40=AM2-47>CGRP. At this receptor AM had a significantly higher Emax than AM2-40 and AM2-47. AM2-40 had a significantly higher Emax than AM2-47 at the AM1 receptor, though the difference was very small (Figure 2, Table 1). At the AM2 receptor the relative rank order of potency was AM=AM2-40=AM2-47>CGRP, there were no significant differences in Emax at this receptor (Figure 2, Table 1). Relative rank orders of potency derived in this study align with the literature4, confirming the validity of our cells and receptor constructs. The partial agonism of AM2-40 and AM2-47 at the AM1 receptor aligns with previously reported literature1, 25, 26. To the authors’ knowledge, our data are the first direct pharmacological comparison of AM2-40 and AM2-47 at defined receptors. In our transfected cell models the two peptide lengths appear equipotent, at least in terms of cAMP production.


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Biochemistry

Figure 2. cAMP production in response to AM2-40, AM2-47, CGRP and AM at the human CGRP, AM1 and AM2 receptors. Data are the mean ± s.e.m. of combined data from 3-9 independent experiments. Table 1. Summary of pEC50 and Emax values for AM, AM2, and αCGRP at the CGRP, AM1, and AM2 receptors. CGRP Receptor

AM1 Receptor

AM2 Receptor

Peptide

pEC50

Emax

n

pEC50

Emax

n

pEC50

Emax

n

AM2-40

9.00 ± 0.05

100

8

8.50 ± 0.08

100

9

9.28 ± 0.14

100

7

AM2-47

8.56 ± 0.10

103.6 ± 7.9

4

8.37 ± 0.12

89.7 ± 1.0*

4

9.19 ± 0.17

98.6 ± 13.4

3

AM

8.02 ± 0.23*

105.5 ± 20.7

4

9.29 ± 0.10*

180.1 ± 26.0*

5

9.82 ± 0.01

74.2 ± 4.1*

3

CGRP

9.88 ± 0.11*

128.5 ± 29.1

4

6.56 ± 0.14*

101.5 ± 18.7

4

6.83 ± 0.31*

102 ± 14.1

3

Values are means ± s.e.m. The potency of peptides were compared within a receptor using one-way ANOVA with Tukey’s multiple comparisons test, significance accepted at p < 0.05. Emax values presented as values normalized to the Emax of AM2-40. Differences in Emax were analyzed using 95% confidence intervals, significance was accepted when 95% confidence intervals did not overlap. * represents a significant difference between AM2-40 and the named peptide. Roh et al (2004) present the only other direct pharmacological comparison of AM2-40 and AM2-47. In their study, AM2-40 was reported to be tenfold more potent at stimulating cAMP production than AM2-47 in SK-N-MC and rat L6 cells, though both peptides were able to compete with radiolabeled CGRP for binding sites on these cells to similar extents1. SK-N-MC cells express human CLR:RAMP1, while rat L6 cells express predominantly CLR:RAMP1 with relatively lower levels of an AM receptor, though this AM receptor is not linked to cAMP production and it is not known whether this is an AM1 receptor, AM2 receptor, or a combination of the two27-29. It is possible that this discrepancy in our findings arises due to differences in receptor expression levels between our transfected cell models and the SK-N-MC or rat L6 skeletal muscle cells which endogenously express receptors30. It is also possible that the complement of intracellular proteins differs between the cell lines, leading to differences in the regulation of downstream signaling pathways25, 30.

Residues within the disulfide loop display differential functional importance in stimulating cAMP production. Given that the two peptides appeared functionally equivalent, alanine substituted analogs were created using AM2-40 as the peptide backbone sequence. The ability of analogs to stimulate cAMP production was compared to AM2-40 at the three CLR based receptors (Figure 3). Broadly speaking, analogs had a similar effect across all tested receptors. That is, a change in signaling ability at the CGRP receptor was matched with a change in signaling at the AM1 and AM2 receptors.


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AM2-40 V11A had a trend for an increased Emax at all tested receptors, however this was not statistically significant. There were no statistically significant differences in the potency of AM2-40 and AM2-40 V11A (Figure 3, Table 2). This is not surprising as valine and alanine are very similar amino-acids, differing by only two methyl groups. This lack of effect with the V11A substitution mirrors results from previous experiments performed using CGRP, where the corresponding residue is an aspartic acid. Substitution of this aspartic acid with cysteine resulted in a small decrease in binding affinity, but no significant difference in cAMP signaling31. Additionally, the aspartic acid in this position can be acetylated, photo-affinity labelled, or tagged with a fluorescent moiety with only minor effects on cAMP signaling32-34. These findings are surprising as in the recently published cryo-EM structures of the CGRP-bound CGRP receptor and the sCTbound CTR this residue is in close proximity to extracellular loop 3 (ECL3)12, 35. Overall, the close proximity of this residue to ECL3 does not necessarily predict an important role, at least in generating receptor conformations that drive cAMP production. AM2-40 L12A had reduced potency and Emax at the CGRP and AM2 receptors, and was unable to stimulate a cAMP response through the AM1 receptor (Figure 3, Table 2). This effect is similar to the effect seen when substituting the equivalent residue in AM. AM F18A has reduced Emax at the AM1 receptor, while remaining a full agonist at the AM2 receptor36. When AM F18 was compromised through a peptide stapling technique, the resulting peptide again had a reduced Emax at the AM1 receptor, but maintained full agonism at the CGRP receptor20. Additionally, the equivalent substitution in CGRP (T4A) causes a reduction in cAMP potency but does not affect Emax at the CGRP receptor31. Thus it seems that the residue in this position is important for stimulating cAMP production through the AM1 receptor, but has less impact at other CLR based receptors. This has been explained mechanistically by Watkins et al. who predict that AM F18 has more direct contacts with CLR ECL3 in the AM1 receptor compared to the AM2 receptor36. However, receptor structures of CLR in complex with RAMP2 and RAMP3 are required to support this explanation.


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CGRP receptor 150

100

-7

-6

-5

-8

-7

-6

-5

0

Log [peptide] M 200

100 50 0

200

AM2-40 AM2-40 L12A

150

-8

-7

-6

-5

cAMP production (% AM2-40)

150 100 50

-12 -11 -10 -9

-8

-7

-6

0

150

50

-8

-7

-6

-5

Log [peptide] M AM2-40 AM2-40 T14A

-12 -11 -10 -9

0


100 50 0

150

-8

-7

-6

-5

0

-12 -11 -10 -9

-8

-7

-6

-5

-5

100 50

-12 -11 -10 -9

-8

-7

-6

-5

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-6

-5

Log [peptide] M AM2-40 AM2-40 T14A

200

AM2-40 AM2-40 T14A

0

0

-6

50

Log [peptide] M 200

-7

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-12 -11 -10 -9

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-12 -11 -10 -9

Log [peptide] M AM2-40 AM2-40 G13A

200

150

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-5

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-5

Log [peptide] M AM2-40 AM2-40 G13A

200

AM2-40 AM2-40 G13A

-6

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100

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-12 -11 -10 -9

-8

AM2-40 AM2-40 L12A

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-8

Log [peptide] M AM2-40 AM2-40 L12A

200

150

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0 -12 -11 -10 -9

0

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0

AM2-40 AM2-40 V11A

150

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AM2 receptor

200

AM2-40 AM2-40 V11A


150

AM1 receptor

200

AM2-40 AM2-40 V11A



200


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Biochemistry

150 100 50 0

0

-12 -11 -10 -9

Log [peptide] M

-8

-7

-6

-5

Log [peptide] M

0

-12 -11 -10 -9

-8

Log [peptide] M

Figure 3. cAMP production in response to AM2-40 and its alanine analogs at the human CGRP, AM1 and AM2 receptors. Data are the mean ± s.e.m. of combined data from 3-4 independent experiments.


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Table 2. Summary of pEC50 and Emax values for AM2-40 analogs at the CGRP, AM1, and AM2 receptors. CGRP Receptor pEC50

AM1 Receptor Emax

WT

Analog

AM2-40 V11A

9.04 ± 0.11

8.89 ± 0.16

132.3 ± 19.3

AM2-40 L12A

9.02 ± 0.06

7.90 ± 0.08*

AM2-40 G13A

9.08 ± 0.09

AM2-40 T14A

9.01 ± 0.11

n

AM2 Receptor

pEC50

Emax

WT

Analog

4

8.44 ± 0.14

8.99 ± 0.41

125.3 ± 5.9

42.0 ± 7.3*

3

8.68 ± 0.07

NC

9.52 ± 0.17

135.4 ± 4.7*

3

8.68 ± 0.11

7.73 ± 0.02*

45.1 ± 6.9*

3

8.71 ± 0.09

n

pEC50

Emax

n

9.39 ± 0.16

123.6 ± 15.4

4

9.18 ± 0.08

8.39 ± 0.08*

75.5 ± 2.7*

3

3

9.27 ± 0.07

9.66 ± 0.09*

121.5 ± 11.1

3

3

9.10 ± 0.15

8.12 ± 0.11*

44.8 ± 6.1*

3

WT

Analog

3

9.24 ± 0.11

NC

4

9.08 ± 0.08*

163.7 ± 18.1

NC

NC

Values represent the mean ± S.E.M. of n independent experiments. The potency of analogs to stimulate cAMP production at CLR based receptors was compared to unmodified AM2-40 using unpaired t-tests, significance accepted at p < 0.05. Differences in Emax between analog and unmodified AM2-40 were considered significant when the 95% confidence intervals did not overlap. * indicates a significant difference. NC used where no curve could be fit to the data.


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Biochemistry

AM2-40 G13A was significantly more potent than AM2-40 at the AM1 and AM2 receptors, and had a trend for an increase in potency at the CGRP receptor (Figure 3, Table 2). The increases in potency were similar across the receptors (~3-fold increase in potency). There were also trends for AM2-40 G13A to have an increased Emax at all tested receptors, though the only case where the difference was significant was at the CGRP receptor. The increase in Emax was largest at the AM1 receptor (Figure 3, Table 2). There was no direct pharmacological comparison performed for AM and AM2-40 G13A, however it does appear that AM2-40 G13A may have a similar Emax to AM at the AM1 receptor, suggesting that this analogue would be a full agonist at this receptor (Table 1, Table 2). This position seems to favor a small residue across the CGRP/CT peptide family (being glycine in AM and AM2, alanine in CGRP and amylin, and serine in CT)4. As the native residue in CGRP is alanine, it is perhaps not surprising that AM2-40 G13A has a slight increase in potency and Emax at this receptor, however to see this small increase across all receptors is more difficult to explain. It is possible that the extra methyl group present on alanine allows for increased contact between the residue and TM6/ECL3 as this is where the residue projects in cryo-EM structures of the CGRP-bound CGRP receptor and the sCT-bound CTR12, 35. Alternatively, having an alanine in this position could enhance an N-capping motif, stabilizing the Nterminus of the peptide in an activating conformation37. The role of this residue has been investigated in amylin and CGRP. In both these peptides, the corresponding residue is natively alanine. An amylin analog which substitutes the native alanine for glycine has reduced potency at cAMP through CTR and the AMY1 receptor (CTR:RAMP1 complex)24, while an analog of CGRP which substitutes the native alanine for cysteine has reduced potency at the cAMP pathway in both Cos7 cells transfected with CTR/RAMP1 and in SK-N-MC cells which natively express CGRP receptors31. It is interesting to note that the cysteine-substituted CGRP analog (CGRP A5C) has lower affinity for CGRP receptors than CGRP8-37, a peptide fragment which omits the disulfide loop entirely31. In contrast, an amylin analog which substitutes the alanine in this position with serine (as found natively in calcitonin) has increased potency at CTR based receptors24. Thus it seems this position is conserved as a small residue across the peptide family, and that this residue has an important role in peptide activity, possibly by maintaining the required orientation of the disulfide loop region. AM2-40 T14A had reduced potency and Emax at the CGRP and AM2 receptors, and was unable to stimulate cAMP production through the AM1 receptor (Figure 3, Table 2). The effect on Emax was consistent between receptors, but the reduction in potency was twice as large at the AM2 receptor than at the CGRP receptor. This residue is conserved across the entire peptide family, and also across species4, 38. Thus, it is not surprising that modifying this residue caused a large reduction in AM2-40 activity. The loss in activity mirrors previous experiments using both amylin and CGRP, in which the threonine in this position was replaced with alanine 24, 31. In both cases the resulting peptide had impaired potency at all tested receptors. There have been no reports where the resulting analog completely loses the ability to elicit cAMP production (as seen here at the AM1 receptor), but there is often a trend for the analog to have a reduced Emax, and the lack of response at the AM1 receptor may reflect an exaggeration of this effect. In the recent cryo-EM structure of the CGRP-bound CGRP receptor, the equivalent threonine (CGRP T6) makes key, long-lasting hydrogen bonds with CLR H29512. Similarly, calcitonin T6 makes key, long lasting interactions with the corresponding residue on CTR (residue H302)35, 39. Additionally, in the CGRP receptor structure, the methyl group of CGRP T6 appears to make contacts with the receptor. A serine substituted analogue of CGRP (CGRP T6S) which retains the hydrogen bond forming capabilities of T6 but lacks the methyl group is less potent than unmodified CGRP, but more potent than CGRP T6A31. This suggests

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the reduction in cAMP signaling associated with AM2-40 T14A is due to a combination of losing the ability to hydrogen bond with CLR H295, as well as losing interactions with other residues on the receptor. In summary, the N-terminal region of AM2-40 appears to play an important role in stimulating cAMP production through CLR based receptors. Recent evidence suggests that the C-terminal region of AM2 appears to interact with isolated ECDs of CLR:RAMP complexes using a different mechanism to CGRP and AM13, 40, however our data shows that there is a conserved mechanism across the peptide family for the peptide N-terminus to interact with the ECLs and TM bundle to stimulate cAMP production. This structure-function insight will guide future drug discovery efforts aimed at developing drugs based on AM2.

Conclusions We report that AM2-40 and AM2-47 are pharmacologically equivalent across all CLR based receptors in transfected Cos7 cells. We also highlight the differential importance of individual amino-acids within the disulfide loop of AM2-40 and produce the first insights into its mechanisms of receptor activation.

ACCESSION CODES CLR (CALCRL) Q16602 RAMP1 O60894 RAMP2 O60894 RAMP3 O60896 ASSOCIATED CONTENT Supporting Information Supplemental Methods – Chemical synthesis of AM2-40 and analogs.

AUTHOR INFORMATION Corresponding Author * Debbie L Hay. E-mail: [email protected]. * Paul W Harris. E-mail: [email protected]

Author Contributions This manuscript was written with the contribution of all authors. H.M. and E.R.H performed experiments. M.A.B. and P.W.R.H. synthesized AM2 analogs. M.L.G., H.A.W., and D.L.H. interpreted experiments. M.L.G., P.W.R.H., and D.L.H. wrote the paper.


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Biochemistry

Funding Sources E.R.H is supported by the Barbara Basham Doctoral Scholarship administered by the Auckland Medical Research Foundation and Perpetual Guardian. M.L.G. is supported by a Health Research Doctoral Scholarship from the University of Auckland. D.L.H was supported by a James Cook Research Fellowship from the Royal Society of New Zealand. P.W.H, D.L.H, and M.A.B are supported by Marsden Grants from the Royal Society of New Zealand. Peptides were synthesized with the support of the Maurice Wilkins Centre for Molecular Biodiscovery.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT ABBREVIATIONS AM, adrenomedullin; AM2, adrenomedullin 2; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CT, calcitonin; ECD, extracellular domain; ECL, extracellular loop; GPCR, G protein-coupled receptor; RAMP, receptor activity-modifying protein.

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For Table of Contents Use Only Adrenomedullin 2 N-terminus T

Q

A

Q

L

L

R

V

G Can be removed C Critical disulfide bond V Substitution not tolerated Substitution tolerated L Substitution improves activity


C T G

15

Pharmacological Characterization and Investigation of N-Terminal

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