Receptor Activity Modifying Proteins Have Limited Effects on the Class

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Receptor activity-modifying proteins have limited effects on the class B G protein-coupled receptor calcitonin receptor-like receptor stalk Michael L Garelja, Christina A Walker, Andrew Siow, Sung H Yang, Paul William R. Harris, Margaret A. Brimble, Harriet A Watkins, Joseph J Gingell, and Debbie L. Hay Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01180 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Biochemistry

Receptor activity-modifying proteins have limited effects on the class B G protein-coupled receptor calcitonin receptor-like receptor stalk Michael L. Garelja1, Christina A. Walker1, Andrew Siow1,2, Sung H. Yang1,2, Paul W.R. Harris1,2, Margaret A. Brimble1,2,3,, Harriet A. Watkins1, Joseph J. Gingell1, Debbie L. Hay1,2*. 1. School of Biological Sciences, 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 *Corresponding author: Debbie L Hay, Mail: [email protected], Tel: +64 9 373 7599

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ABBREVIATIONS AM – adrenomedullin, CGRP – calcitonin gene-related peptide, CLR – calcitonin receptor-like receptor, CT - calcitonin, ECD – extracellular domain, ECL- extracellular loop, GLP-1 – glucagon-like peptide-1, GPCR – G protein-coupled receptor, RAMP – receptor activity modifying protein, TM - transmembrane


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Biochemistry

ABSTRACT

The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) that forms the basis of three pharmacologically distinct receptors, the calcitonin gene-related peptide (CGRP) receptor, and two adrenomedullin (AM) receptors. These three receptors are created by CLR interacting with three receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids which is referred to as the “stalk”. Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP production, and in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all receptors. N128A, V135A and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other receptor regions.

KEYWORDS G protein-coupled receptor, Receptor activity-modifying proteins, calcitonin receptor-like receptor, adrenomedullin, calcitonin gene-related peptide


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INTRODUCTION G protein-coupled receptors (GPCRs) are one of the largest families of proteins in the human body. GPCRs convert extracellular stimuli into intracellular responses. GPCRs are involved in a diverse range of physiological processes and a large number of drugs on the market target these receptors1. Class B GPCRs are a subset of GPCRs, which bind peptide hormones as their endogenous ligands. These receptors have distinct domains, each of which performs a specific role. The Nterminal extracellular domain (ECD) binds the C-terminus of the peptide hormone, facilitating interactions between the N-terminal region of the peptide and the extracellular portion of the transmembrane (TM) bundle. The ECD is joined to the TM bundle by a flexible stretch of amino acids, often termed the “stalk”2. This stalk region may act as a hinge3. The TM bundle undergoes a conformational change between inactive and active states, and the intracellular region is involved in interactions with G proteins and other intracellular proteins to cause an intracellular response4. However, this model is likely an over-simplification of reality, as the ECD is reported to play a role in signal transduction in a number of class B GPCRs5, perhaps transmitted through the stalk to the TM bundle or through contacts between the ECD and extracellular loops (ECL). Structural studies have yielded valuable insights into the structure of ECDs6-12 and TM domains2, 13-15

of class B GPCRs, however until recently structures of these domains had only been

determined independently, or together at a low resolution3. This is in part due to the apparent flexibility of the stalk, which allows the ECD to adopt multiple different conformations relative to the TM domain

3, 16, 17

, with the receptor being capable of adopting a closed conformation

stabilized by interactions between the ECD and TM domain 3, 18.


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Biochemistry

Newly published structures have captured the ECD and TM domains together, providing insights into conformational states. There are full length crystal structures of the glucagon receptor in complex with an allosteric antibody and with a glucagon analog bound19,20, an active peptidefragment bound glucagon-like peptide-1 (GLP-1) receptor17, and cryo-electron microscopy structures of the peptide bound active calcitonin (CT)16 and GLP-1 receptors21. In the inactive glucagon receptor structure, the stalk region is in an extended β-sheet formation, forming multiple interactions with ECL1, potentially stabilizing the inactive state of the receptor20. In the recent structure of the glucagon receptor bound to a glucagon analog, conformational changes were observed in the stalk and ECL1, with the stalk forming a three helical turn extension of the TM1 α-helix with ECL1 no longer associating with the stalk, and instead being located above TMs 2&3. The position of the ECD relative to the TM domain is shifted ~ 90° compared to the antibody-bound structure19. This position of the ECD is also observed in the cryo-EM structure of the GLP-1 receptor, however the stalk region was only partially resolved in this structure21. In the cryo-EM structure of the CT receptor neither the ECD nor the stalk were resolved16, highlighting the flexibility associated with this region. In the crystal structure of the active GLP1 receptor bound to a truncated peptide agonist the stalk has less secondary structure than noted in other crystal structures, but makes a number of contacts with the ECD, ECL1, and the peptide. It is possible that this difference results from the study using a truncated peptide which makes fewer contacts with the stalk and ECD than a full length peptide17. The two crystal structures of the full length glucagon receptor19, 20 along with previous functional studies provide evidence that the stalk may act as a hinge, allowing the ECD to interact with ECL 3 to stabilize the inactive conformation of the receptor18. The flexibility of the stalk, coupled with the inherent limitation of crystal structures (in that they only provide a snapshot of


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a dynamic entity) means that functional studies of the region are important to fully understand its role in class B GPCR activity. In addition to allowing the ECD to adopt multiple conformations there is evidence that in class B GPCRs the stalk plays a role in ligand binding and receptor activation3,

19, 22-26

, as well as influencing receptor stability and cell surface expression22,

23

.

Overall, the structure and function of the “stalk” sequence of amino acids that is found in all class B GPCRs is still unclear. Matters are complicated by the ability of several class B GPCRs to interact with receptor activity-modifying proteins (RAMPs)27. In mammals there are three RAMPs which contain a long ECD, a single pass TM domain, and a short intracellular tail27. Their interactions are often studied with the calcitonin receptor-like receptor (CLR) because CLR is an obligate dimer with a RAMP. The three human RAMPs interact with CLR to create three pharmacologically distinct receptors, the calcitonin gene-related peptide (CGRP) receptor (CLR/RAMP1), and two adrenomedullin (AM) receptors, the AM1 receptor (CLR/RAMP2) and the AM2 receptor (CLR/RAMP3)27. Crystal structures of CLR/RAMP ECD complexes suggest that part of RAMP function is a direct interaction with the receptor ECD and the peptide8, 10, 28, 29, but evidence from functional studies suggest that RAMPs can also influence other receptor regions such as ECLs30, the network of residues in the TM bundle which contributes to receptor activation31, as well as intracellular G protein-coupling32,

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. At present there is little information regarding whether

RAMPs interact with the GPCR stalk, and if so, whether RAMPs are able to alter the conformation of this region to have a stabilizing effect on this otherwise flexible region. It is also not known whether the CLR stalk might provide contacts for its endogenous peptide ligands, such as the two calcitonin gene-related peptides (α and βCGRP) or adrenomedullin (AM).


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Biochemistry

Hence the goal of this study was to investigate the CLR stalk to determine the functional contribution of individual amino acids within this region. We performed an alanine scan of CLR from residues T125 to T145, excluding the alanine within this sequence (Figure 1). We selected a few residues within the ECD, starting at T125. This was selected as the starting point, rather than Y124 because Y124 is known to form part of the ligand binding pocket in both CGRP and AM receptors, and has also previously been investigated through alanine mutagenesis10, 28. We progressed through the stalk to T145, which was chosen as the last residue based on models of CLR, where T145 appears to be two α-helical turns within TM130, 31. Given the likely flexibility of this region, its length could be different in distinct activation states. Figure 1 shows this region aligned with the equivalent region of other class B GPCRs. CLR mutants were tested with all three RAMPs, with effects measured through cell surface expression, cAMP signaling, and in select cases, radioligand binding and total cell expression. This research gives insight into the CLR stalk, and whether RAMPs affect this function.


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Figure 1. A) Model of the active peptide bound AM1 receptor (CLR/RAMP2)30. CLR is green, RAMP2 is violet, Gαs fragment is yellow, AM is dark blue, and the stalk region studied here is orange. Image created in Pymol. B) Amino acid sequence alignment of the stalk region of class B GPCRs. The sequences were aligned in Geneious v 10.05 using the Geneious alignment function. Residues highlighted in Green represent 100%, yellow 60%-80% and unhighlighted less than 60% similarity.


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Biochemistry

MATERIALS AND METHODS Materials-Human AM was from Bachem (Bubendorf, Switzerland). Human αCGRP was synthesized in house as previously described34. Human βCGRP was synthesized in house (detailed methods in Supporting Information). LANCE cAMP assay kits, reagents, plates, and [125I]-human iodohistidyl10-αCGRP were purchased from PerkinElmer (Waltham, MA, USA). Expression constructs and mutagenesis-Human CLR with an N-terminal hemagglutinin (HA) epitope tag was used for all experiments. Human RAMP 1 with an N-terminal myc epitope and human RAMP2 with an N-terminal FLAG epitope were used in experiments. HA-CLR, mycRAMP1 and FLAG-RAMP2 have been shown to have comparable pharmacology to wildtype (WT) RAMP1 and RAMP235, 36. Untagged human RAMP3 was also used in this study37. HACLR was mutated by a method based on the Quik Change II site-directed mutagenesis kit (Stratagene, Cambridge, UK) as described previously38,

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. Primers encoding mutations were

generated using PrimerX (www.bioinformatics.org/primerx) and custom synthesized by Integrated DNA Technologies (IDT, IA, U.S.A). Plasmid DNA was extracted using NucleoBond Xtra Maxiprep kit with protocol described by manufacturer (Macherey-Nagel, Duren, Germany). Mutations were confirmed through sequencing (Genomics Centre, Auckland Science Analytical Services, The University of Auckland, Auckland, New Zealand). Cell culture and transfection-Cos7 cells were cultured as previously described as they do not endogenously express CLR or RAMPs40.Cells used were between passage 18 and 30. Cells were cultured in DMEM with 8% heat-inactivated fetal bovine serum and grown in a 37°C humidified incubator with 95% air and 5% CO2. Cells were seeded at 20,000 cells per well (as determined by a Countess CounterTM, Invitrogen, Carlsbad, CA, USA) in 96 well Spectraplates


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(PerkinElmer) for cAMP or ELISA experiments, 96 well CellCarrier plates (PerkinElmer) for immunofluorescence experiments, or at 50,000 cells per well in 48 well Falcon Polystyrene Microplates (ThermoFisher Scientific) for radioligand binding experiments. Cells were transfected 1 day after plating using polyethylenimine using a 1:1 ratio of CLR to RAMP DNA40. Cells were left in transfection mix for two days before experiments. Experimental Design-All experiments were independently replicated at least three times with triplicate or quadruplicate technical replicates, depending on the experiment. A WT control was included in each experiment, and mutants were statistically compared to only those controls, not the mean of all WT controls, giving equal n between WT and mutants. Cell Surface Expression ELISAs-Cell surface expression of HA-CLR was measured through an ELISA protocol as previously described38,

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with modifications. Rather than adding 8%

paraformaldehyde (PFA) in phosphate buffered saline (PBS) to wells containing media (final volume 4 % PFA), wells were aspirated and 100 µL of 4% PFA in PBS was added. Goat serum in (10% in PBS) was used as a blocking agent. Mouse anti-HA (50 µL, Sigma H-9658) was diluted 1:2000 in 1 % goat serum in PBS and was used as a primary antibody; the plate was incubated at 37°C for 30 minutes. The secondary antibody was goat anti-mouse antibody conjugated to horseradish peroxidase (Sigma A-4416, 50 µL diluted 1:2000 in 1 % goat serum in PBS) which was incubated for 1 hour at room temperature. The rest of the protocol was unchanged. Total Cell Expression ELISAs-Total cell expression was measured though an ELISA protocol as above but 0.2% Triton X-100 in PBS was used as a replacement for PBS for all steps except for


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Biochemistry

antibody dilutions, the wash step between primary and secondary antibody additions, and the post cresyl-violet wash step. Immunofluorescence-Cell surface and total cell expression immunofluorescence experiments were performed essentially as above for ELISAs. The initial H2O2 step was omitted and the secondary antibody used was goat anti-mouse conjugated to Alexa 594 (ThermoFisher Scientific, A-11032 diluted in 1:400 in 1 % goat serum in PBS). After the secondary antibody was incubated at room temperature for 1 hour, the plate was washed once with 100 µL PBS. DAPI (ThermoFisher Scientific, D1306) was diluted to 300 nM in PBS and 50 µL added to each well. Cells were incubated for 5 minutes at room temperature, the plate was then washed once with 100 µL PBS. Wells were visualized in 100 µL PBS using an Operetta high content imager (PerkinElmer, HH12000000) using excitation filters 360-400 nm and 560-580 nm, and emission filters of 410-480 nm and 590-640 nm at 20x and 40x magnification. cAMP assay-Cells were stimulated with agonists in triplicate as previously described31, 40 with the incubation of LANCE detection mix extended from 1 hour to 4 hours. Plates were read using an EnVision microplate reader (PerkinElmer, MA, USA) or a BMG Clariostar plate reader (BMG Labtech, Germany). Radioligand binding-Radioligand binding experiments were performed as described previously41. Briefly, on the day of experiments the cells were washed once with warm binding buffer (comprised of DMEM + 0.1 % BSA), which was then removed and replaced with warm binding buffer.

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I-αCGRP was added at approximately 30,000 cpm per well. After radiolabel

was added either binding buffer (total binding) or 3 µM αCGRP (non-specific binding) was added to the relevant wells. The plate was left to incubate at room temperature for one hour.


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Media was then removed and cells washed once with ice-cold PBS before solubilization with 0.2 M NaOH for five minutes. The resulting lysate was transferred to tubes and read on a Wizard2 gamma counter (PerkinElmer). Data analysis for ELISAs-Experimental values were calculated as absorbance at 490 nm minus absorbance at 650 nm. The resulting value was then divided by absorbance at 595 nm. Individual experiments were normalized using the mean value of vector control (PCDNA) as 0 % and the mean value of WT CLR/RAMP as 100 %. Combined results were analyzed in GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) and statistical significance was achieved if the 95% confidence intervals did not include 100% (WT) expression. This analysis method was used as normalization caused WT expression to always be 100%. Data analysis for immunofluorescence-Data analysis was performed using Columbus software (PerkinElmer, MA, USA). For data analysis, at least 4 fields of view (taken at 20x) were analyzed for each well; the positions of these fields was consistent between wells. The sum of Alexa-594 intensity in each well was calculated. Values were normalized using the mean of Alexa-594 intensity in WT CLR/RAMP1 wells as 100 % and the mean of Alexa-594 intensity in PCDNA control wells as 0 %. Combined results were analyzed in GraphPad Prism and statistical significance was achieved if the 95% confidence intervals did not include 100% expression (WT). Data analysis for cAMP assays-Data analysis was performed in GraphPad Prism 7. 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 comparing WT and mutant pEC50 values were


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Biochemistry

analyzed by unpaired t-tests. t-tests were used for analysis because experiments involved a WT receptor and a random three mutants on each 96 well plate. This essentially created a paired experimental design, where mutant results were linked to the results of the WT receptor on the same plate in each independent experiment but not to other mutants. Potency differences between different peptides at WT receptors were analyzed by one-way ANOVA because each independent experiment had all peptides together on each plate. Normalized Emax values were obtained by normalizing data from each experiment to the fitted maximum and minimum of the WT control in each experiment. Statistical significance was achieved if the 95% confidence interval did not include 100%. Normalized data sets were generated by combining the mean of data points from each individual experiment. Data analysis for radioligand binding-Specific binding in each experiment was determined by subtracting the mean of non-specific binding at individual receptors from the total binding at each corresponding receptor. Individual experiments were then normalized using the mean WT value as 100%. Combined results were analyzed in GraphPad Prism 7 and statistical significance was achieved if the 95% confidence intervals did not include 100%.

RESULTS Effect of alanine substitution on CLR cell surface expression -There was no significant change to cell surface expression of CLR alanine mutants compared to WT with the exception of C127A, N140A, F142A and L144A (Tables 1 and 2, Figure 2). These mutations caused a decrease in cell surface expression to between ~3 and ~55% of WT expression with all three RAMPs in ELISAs. For RAMP1, these findings were confirmed through the use of


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immunofluorescence (Figure 2). These mutants were investigated further through total cell expression ELISAs. The mutations N140A, F142A and L144A caused a ~50 to ~70% decrease in total cell expression relative to WT in ELISAs. These findings were also confirmed through immunofluorescence (Figure 2). The total cell expression of C127A was not significantly different

from

WT

through

either

detection

method.


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Biochemistry

Figure 2. A) Comparison of cell surface expression (CSE) and total cell expression (TCE) for selected CLR mutants expressed with RAMP1, as analyzed by ELISA and immunofluorescence (IF) protocols. Bars are mean ± S.E.M. of three independent experiments performed in triplicate * p < 0.05 if 95% confidence intervals did not include 100% (WT) expression. Representative immunofluorescence results for B) cell surface expression and C) total cell expression. Images taken using a 40xWD lens and are representative of 3 individual experiments performed in quadruplicate. Scale bar indicates 50 µM. DAPI is blue and CLR is orange.

Effect of alanine substitution on AM-stimulated cAMP production- AM acts as an agonist at all three CLR/RAMP complexes42 (Figure 3), thus mutations were tested with AM at all three


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receptors. The majority of mutations had only small effects on AM-stimulated cAMP production, with less than 3-fold reductions in pEC50 (Table 1 and 2, Figure 4). Mutant Emax values were typically within 30% of WT (Table 1 and 2). There was no effect on basal signaling with any mutant as is evident from curves (Figures 4-6).

Figure 3. Concentration-response curves for human AM, αCGRP and βCGRP at the human CGRP, AM1 or AM2 receptors (upper panel). Each point is the mean ± S.E.M. of 3 independent experiments performed in triplicate. The bottom panel shows scatter plots of these data, with each point being the pEC50 from each individual experiment that was used to generate the mean data in the upper panel. Data are the mean ± S.E.M. Data analyzed by one way ANOVA with post-hoc Dunnett’s test. * p < 0.05 vs. αCGRP.


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Biochemistry

Figure 4. Concentration-response curves for mutants that had little impact on cAMP signaling at any ligand/receptor combination. Each point is the mean ± S.E.M. of at least 3 independent experiments performed in triplicate. At the CGRP receptor, K134A caused a small (~3-fold) reduction in AM potency, however this effect was not significant at the AM receptors. Similarly, at the AM1 receptor V135A caused a small (~3-fold) reduction in AM potency; this reduction in AM potency was not significant at the other receptors. At the AM2 receptor, T131A caused a small (5-fold) reduction in AM potency. At the CGRP and AM1 receptors comprised of CLR T131A there was a trend for a reduction in AM potency, but this was not statistically significant (Tables 1 and 2, Figure 4 and 5).


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On the other hand T125A caused a large (>10-fold) decrease in AM potency at the CGRP and AM2 receptors (Tables 1 and 2) and a smaller (~4-fold) decrease at the AM1 receptor (Tables 1 and 2, Figure 5). Receptors comprised of CLR C127A, N140A, F142A or L144A failed to produce cAMP in response to AM (Figure 5). These findings are not surprising given the greatly reduced cell surface expression of these mutants.


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Figure 5. Concentration-response curves for mutants that had similar impacts on cAMP signaling across all receptor/ligand combinations. Each point is the mean ± S.E.M. of at least 3 independent experiments performed in triplicate.


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Effect of alanine substitution on αCGRP-stimulated cAMP production –αCGRP is a potent agonist at CLR/RAMP1, however it is much weaker at other CLR based receptors42 (Figure 3). Hence, αCGRP was only tested at CLR/RAMP1. The majority of mutations had only small effects on pEC50 (

Receptor Activity Modifying Proteins Have Limited Effects on the Class

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