Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking

David N. Langelaan; Tyler Reddy; Aaron W. Banks; Graham Dellaire; Denis J. Dupré; Jan K. Rainey

doi:10.1016/j.bbamem.2013.02.005

Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking

David N. Langelaan, Tyler Reddy, Aaron W. Banks, Graham Dellaire, Denis J. Dupré, Jan K. Rainey

Medicine

Research output: Contribution to journal › Article › peer-review

40 Citations (Scopus)

Abstract

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55 ^1.59. Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46^1.50 is central to a disruption in helical character. G42^1.46, G45^1.49 and N46^1.50, which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

Original language	English
Pages (from-to)	1471-1483
Number of pages	13
Journal	Biochimica et Biophysica Acta - Biomembranes
Volume	1828
Issue number	6
DOIs	https://doi.org/10.1016/j.bbamem.2013.02.005
Publication status	Published - Jun 2013

Bibliographical note

Funding Information:
This research was supported by Canadian Institutes of Health Research (CIHR) Operating Grants to JKR ( MOP-111138 and ROP-91807 ) and GD ( MOP-84260 ) and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to DJD ( RGPIN/355310-2008 ). ROP-91807 was partnered through the Nova Scotia Health Research Foundation and Dalhousie University . DNL was the recipient of a Canada Graduate Scholarship (CGS) from NSERC ; TR was previously supported by an NSERC CGS and a Killam Predoctoral Scholarship and is currently supported by a CIHR Postdoctoral Fellowship ; AWB was the recipient of a CGS from the CIHR and a Killam Predoctoral Scholarship; GD, DJD and JKR are supported by CIHR New Investigator Awards ; and, GD is a Senior Scientist of the Beatrice Hunter Cancer Research Institute and the Cameron Research Scientist of the Dalhousie University Cancer Research Program.

Funding Information:
NMR experiments were recorded (1) with support from Dr. Tara Sprules at the Québec/Eastern Canada High Field NMR Facility, supported by NSERC, the Canada Foundation for Innovation (CFI), the Québec ministère de la recherche en science et technologie, and McGill University; and, (2) at the NMR 3 Facility, supported by Dalhousie University, using the 700 MHz housed at the National Research Council of Canada's Institute for Marine Biosciences (NRC-IMB) and a He-cooled probe funded by an Atlantic Canada Opportunities Agency Grant to Dalhousie University. Simulations were performed using resources at Dalhousie University and, predominantly, at the Structural Bioinformatics & Computational Biochemistry Unit, Oxford, U.K. Grants from NSERC, the Dalhousie Medical Research Foundation and the CFI provided equipment essential for this work. We thank Dr. Philip Fowler (SBCB, Oxford, UK) for helpful advice regarding simulations. Finally, we are grateful to Dr. Devanand M. Pinto (NRC-IMB) for providing MALDI-MS access.

ASJC Scopus Subject Areas

Biophysics
Biochemistry
Cell Biology

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10.1016/j.bbamem.2013.02.005

Cite this

@article{fd3b6355caf041e78b716b1f37c4fb3c,

title = "Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking",

abstract = "G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55 1.59. Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N461.50 is central to a disruption in helical character. G421.46, G451.49 and N461.50, which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.",

author = "Langelaan, {David N.} and Tyler Reddy and Banks, {Aaron W.} and Graham Dellaire and Dupr{\'e}, {Denis J.} and Rainey, {Jan K.}",

note = "Funding Information: This research was supported by Canadian Institutes of Health Research (CIHR) Operating Grants to JKR ( MOP-111138 and ROP-91807 ) and GD ( MOP-84260 ) and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to DJD ( RGPIN/355310-2008 ). ROP-91807 was partnered through the Nova Scotia Health Research Foundation and Dalhousie University . DNL was the recipient of a Canada Graduate Scholarship (CGS) from NSERC ; TR was previously supported by an NSERC CGS and a Killam Predoctoral Scholarship and is currently supported by a CIHR Postdoctoral Fellowship ; AWB was the recipient of a CGS from the CIHR and a Killam Predoctoral Scholarship; GD, DJD and JKR are supported by CIHR New Investigator Awards ; and, GD is a Senior Scientist of the Beatrice Hunter Cancer Research Institute and the Cameron Research Scientist of the Dalhousie University Cancer Research Program. Funding Information: NMR experiments were recorded (1) with support from Dr. Tara Sprules at the Qu{\'e}bec/Eastern Canada High Field NMR Facility, supported by NSERC, the Canada Foundation for Innovation (CFI), the Qu{\'e}bec minist{\`e}re de la recherche en science et technologie, and McGill University; and, (2) at the NMR 3 Facility, supported by Dalhousie University, using the 700 MHz housed at the National Research Council of Canada's Institute for Marine Biosciences (NRC-IMB) and a He-cooled probe funded by an Atlantic Canada Opportunities Agency Grant to Dalhousie University. Simulations were performed using resources at Dalhousie University and, predominantly, at the Structural Bioinformatics & Computational Biochemistry Unit, Oxford, U.K. Grants from NSERC, the Dalhousie Medical Research Foundation and the CFI provided equipment essential for this work. We thank Dr. Philip Fowler (SBCB, Oxford, UK) for helpful advice regarding simulations. Finally, we are grateful to Dr. Devanand M. Pinto (NRC-IMB) for providing MALDI-MS access. ",

year = "2013",

month = jun,

doi = "10.1016/j.bbamem.2013.02.005",

language = "English",

volume = "1828",

pages = "1471--1483",

journal = "Biochimica et Biophysica Acta - Biomembranes",

issn = "0005-2736",

publisher = "Elsevier",

number = "6",

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TY - JOUR

T1 - Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking

AU - Langelaan, David N.

AU - Reddy, Tyler

AU - Banks, Aaron W.

AU - Dellaire, Graham

AU - Dupré, Denis J.

AU - Rainey, Jan K.

N1 - Funding Information: This research was supported by Canadian Institutes of Health Research (CIHR) Operating Grants to JKR ( MOP-111138 and ROP-91807 ) and GD ( MOP-84260 ) and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to DJD ( RGPIN/355310-2008 ). ROP-91807 was partnered through the Nova Scotia Health Research Foundation and Dalhousie University . DNL was the recipient of a Canada Graduate Scholarship (CGS) from NSERC ; TR was previously supported by an NSERC CGS and a Killam Predoctoral Scholarship and is currently supported by a CIHR Postdoctoral Fellowship ; AWB was the recipient of a CGS from the CIHR and a Killam Predoctoral Scholarship; GD, DJD and JKR are supported by CIHR New Investigator Awards ; and, GD is a Senior Scientist of the Beatrice Hunter Cancer Research Institute and the Cameron Research Scientist of the Dalhousie University Cancer Research Program. Funding Information: NMR experiments were recorded (1) with support from Dr. Tara Sprules at the Québec/Eastern Canada High Field NMR Facility, supported by NSERC, the Canada Foundation for Innovation (CFI), the Québec ministère de la recherche en science et technologie, and McGill University; and, (2) at the NMR 3 Facility, supported by Dalhousie University, using the 700 MHz housed at the National Research Council of Canada's Institute for Marine Biosciences (NRC-IMB) and a He-cooled probe funded by an Atlantic Canada Opportunities Agency Grant to Dalhousie University. Simulations were performed using resources at Dalhousie University and, predominantly, at the Structural Bioinformatics & Computational Biochemistry Unit, Oxford, U.K. Grants from NSERC, the Dalhousie Medical Research Foundation and the CFI provided equipment essential for this work. We thank Dr. Philip Fowler (SBCB, Oxford, UK) for helpful advice regarding simulations. Finally, we are grateful to Dr. Devanand M. Pinto (NRC-IMB) for providing MALDI-MS access.

PY - 2013/6

Y1 - 2013/6

N2 - G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55 1.59. Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N461.50 is central to a disruption in helical character. G421.46, G451.49 and N461.50, which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

AB - G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55 1.59. Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N461.50 is central to a disruption in helical character. G421.46, G451.49 and N461.50, which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

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Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking

Abstract

Bibliographical note

ASJC Scopus Subject Areas

UN SDGs

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