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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02783

Journal of Cell Science 119, 910-922 (2006)
Published by The Company of Biologists 2006
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Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms

Leonid L. Nikitenko1,2,3,*, Nicola Blucher1, Stephen B. Fox4, Roy Bicknell2, David M. Smith5 and Margaret C. P. Rees1

1 Nuffield Department of Obstetrics and Gynaecology, The University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
2 Molecular Angiogenesis Laboratory, Cancer Research UK, Weatherall Institute of Molecular Medicine, The University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
3 Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, UK
4 Nuffield Department of Clinical Laboratory Sciences, The University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
5 AstraZeneca, CVGI, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK


Figure 1
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  		Fig. 1. Transiently expressed and endogenous hCL species. The human endothelial CL cDNA was cloned into the pcDNA 3.1 expression vector and termed hCLpcDNA. To examine the functionality, the resulting vector was transiently cotransfected with RAMPs into HEK293T cells and the intracellular cAMP concentrations were measured. The intact HEK293T cells lacked functional AM and CGRP receptors, because they showed little cAMP response to agonist stimulation. By contrast, in HEK293T cells cotransfected with hCL-RAMP, the receptors were fully functional in intracellular cAMP production depending on the ligand specificity of the RAMPs (data not shown). (A) Total cell or (B) tissue lysates were analysed by SDS-PAGE under reducing conditions, and immunoblots were probed using polyclonal anti-hCL antibody LN-1436. (A) The antibody specifically recognises hCL in HEK293T cells transfected with hCLpcDNA alone or together with RAMP1 or RAMP2 (CL+RAMP1 or CL+RAMP2; functional CGRP and AM receptors, respectively). HEK293T cells non-transfected (MOCK) or transfected with empty pcDNA vector (pcDNA) or RAMP1 or RAMP2 alone do not express hCL. The ~40-45 kDa hCL species (open diamonds; core-glycosylated receptor) are present in HEK293T cells transfected with hCLpcDNA only. The ~55 kDa hCL species (black diamonds; mature fully glycosylated receptor) is only produced when the receptor is co-expressed with RAMPs. (For details of deglycosylation experiments confirming the origin of hCL species, see supplementary material, Fig. S2.) (B) The antibody also recognises endogenous hCL species expressed in tissues. Arrowheads, deglycosylated (~37 kDa) form of the receptor. The immunoblots are representative of two independent experiments. For loading controls, the membrane was re-probed with an antibody against ß-actin.

 

Figure 2
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  		Fig. 2. Localisation of EndoCL in human tissues. Localisation of hCL was assessed by (A-F) immunohistochemistry on paraffin sections from TMAs using primary LN-1436 and secondary alkaline phosphatase-conjugated antibodies, and detected with Vector Red (red colour). Cell nuclei were counterstained with haematoxylin (blue colour). Note the predominant CL expression in microvascular endothelium in (A) endometrium, (B) myometrium, (C) adenocarcinoma, (D) corpus luteum, and (E and F) cervical stroma (arrows); and in (F) cervical epithelium (arrowheads). (For hCL expression in other tissues, see supplementary material, Figs S3 and S4.)

 

Figure 3
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  		Fig. 3. Expression of endogenous hCL species and RAMP mRNA in endothelial cells. (A) Expression of endogenous hCL species was analysed by immunoblotting. hDMVEC lysates were treated with endoglycosidase F (F, lane 2), endoglycosidase H (H, lane 3) or vehicle (-, lane 1) before SDS-PAGE under reducing conditions and immunoblotting with polyclonal anti-hCL antibody LN-1436. Arrowheads, deglycosylated (~37 kDa); open diamonds, core-glycosylated (~45 kDa); black diamonds, mature fully glycosylated (~55 kDa) forms of the receptor. The ~55 kDa hCL species are reduced to a ~37 kDa hCL band after endoglycosidase F treatment, but are resistant to endoglycosidase H. For loading controls, the membrane was reprobed with an antibody against ß-actin. The immunoblot is representative of two independent experiments. (B) Expression of CL and RAMP mRNAs in primary hDMVECs (lane 1) was analysed by RT-PCR. The set of primers for detection of ß-actin was used as a loading control. RNA sample from kidney (lane 2) served as a positive control. Numbers to the right indicate PCR fragment size. (For details of a full RT-PCR screen of all endothelial cell lines used in the present study, see supplementary material, Fig. S5.)

 

Figure 4
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  		Fig. 4. Subcellular localisation of EndoCL receptor in endothelial cells. (A-D) Intracellular distribution of EndoCL in hDMVECs was assessed by immunofluorescence using LN-1436 antibody and markers of individual cellular structures and organelles: (A) plasma membrane, (B) Golgi, (C) endoplasmic reticulum and (D) lysosomes (see Materials and Methods). The appropriate FITC- (for the detection of hCL; left, first image) or Texas Red- (for the detection of subcellular structures and organelles; centre, second image) conjugated secondary antibodies were used. DAPI was used to counterstain cell nuclei. Colocalized structures (antigens; right, third image) appear in yellow (as indicated by yellow arrows) as determined by overlay of images. Non-colocalised structures appear in green (green arrows) and red (red arrows). Figures are representative of three independent experiments. (For details of a full immunofluorescence screen of intracellular distribution of EndoCL, see supplementary material, Fig. S7.) (E) Localisation of EndoCL at the cell surface in hDMVECs. Reconstructed immunofluorescent confocal pictures of EndoCL in hDMVECs. The en face picture is the collapsed serial of the XY plane confocal image along the Z direction. At the top is the XZ cross-section picture with the apical side facing up and the basal side facing down. To the right of the en face picture is the YZ cross-section picture with apical side on the right and basal side on the left.

 

Figure 5
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  		Fig. 5. Functional endogenous AM and CGRP receptors in endothelial cells. Human DMVECs were seeded into the upper chamber of Transwell inserts. Test peptide [human AM, CGRP and amylin (AMY)] was added to the lower chamber. After 24 hours incubation, the cells were labelled with a fluorescent dye (calcein-AM). Only those labelled cells that have migrated through the pores of the FluoroBlok membrane can be detected. The number of cells migrated to the lower surface of the insert was determined by the measurement of fluorescence of invaded cells in a fluorescence plate reader with bottom reading capabilities. Full microvascular endothelial growth medium (Full Medium) and VEGF were used as positive controls. Each point represents the mean ± s.e.m. of five separate experiments (*P<0.05; **P<0.01; ***P<0.001). (For images, see supplementary material, Fig. S9.)

 

Figure 6
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  		Fig. 6. Dynamics of internalisation and recycling of EndoCL in response to AM and CGRP. Subcellular distribution of EndoCL in hDMVECs before and after exposure to ligand (100 nM; AM or CGRP) for 30 minute. Cells were processed for immunocytochemistry immediately after exposure, or 2 hours after the complete removal of the agonist from the culture medium (see images labelled 120 mins). Colocalisation of EndoCL with the endothelial cell surface (CD31; top row), early sorting endosome (EEA1; middle row) or lysosome (LAMP1; bottom row) markers (see Materials and Methods). Only merged images are presented. Colocalised structures (antigens; yellow arrows) appear in yellow as determined by overlay of the images. Non-colocalised structures appear in green (green arrows) and red (red arrows). The figures are representative of three independent experiments.

 

Figure 7
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  		Fig. 7. Desensitisation of endogenous AM and CGRP receptors in endothelial cells. The dynamics of desensitisation of endogenous AM and CGRP receptors in hDMVECs was investigated using an Akt phosphorylation (Akt-P) assay to determine Akt activity. In control groups, cells were exposed to 100 nM AM (AM, lanes 2-4) or CGRP (CGRP, lanes 11-13) for indicated times. In pretreatment groups (Pretreatment with AM or Pretreatment with CGRP; -/+ indicate if pretreatment was performed), cells were exposed to 100 nM AM (lanes 5-9) or CGRP (lanes 14-18) for 15 minutes, and then to a ligand-depleted medium for a further 30 minutes. In pretreatment groups, cells were then re-challenged with either the same agonist (100 nM) (lanes 5-8 for AM, and lanes 14-17 for, respectively) or reciprocal agonists (100 nM) (CGRP for AM-pretreated cells, lane 9; and AM for CGRP-pretreated cells, lane 18) to examine receptor desensitisation. VEGF-treated cells (10 ng/ml for 10 minutes; lane 10) were used as a positive control.

 

Figure 8
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  		Fig. 8. Interaction of AM and CGRP with endothelial EndoCL. Subcellular distribution of EndoCL in hDMVECs before treatment (Control) after exposure to AM (AM, 100 nM for 15 minutes) with or without antagonists (AM22-52 and CGRP8-37, 1 µM) was assessed by immunofluorescence. Cells were processed for immunofluorescence immediately after exposure. Immunofluorescence was performed using anti-hCL antibody LN-1436 and FITC-conjugated secondary antibody. DAPI was used to counterstain cell nuclei. Figures are representative merged images of two independent experiments. Cell surface (green arrows) and internalised (green arrowheads) receptor portions are indicated.

 

Figure 9
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  		Fig. 9. Properties of endothelial EndoCL and the associated AM/CGRP receptor system. (I) Endothelial EndoCL is expressed on the cell surface as a mature glycoprotein (N-linked carbohydrate residues are schematically represented as grey boxes). Both AM and CGRP induce Akt phosphorylation (Akt-P). (IIA) Receptor is internalised and (III) targeted for degradation upon activation by AM, but (IIB) remains at the cell surface after interaction with CGRP. Interaction with either AM (I-IIA-III) or CGRP (I-IIB) results in (IV) desensitisation of the receptor to both peptides, irrespective of which agonist was used for initial stimulation, and a loss of activity through distinct alternative mechanisms.

 

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© The Company of Biologists Ltd 2006