Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors

Michael L Garelja; Maggie Au; Margaret A Brimble; Joseph J Gingell; Erica R Hendrikse; Annie Lovell; Nicole Prodan; Patrick M Sexton; Andrew Siow; Christopher S Walker; Harriet A Watkins; Geoffrey M Williams; Denise Wootten; Sung H Yang; Paul W R Harris; Debbie L Hay

doi:10.1021/acsptsci.9b00083

Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors

ACS Pharmacol Transl Sci. 2020 Feb 26;3(2):246-262. doi: 10.1021/acsptsci.9b00083. eCollection 2020 Apr 10.

Authors

Michael L Garelja¹, Maggie Au^{1

2}, Margaret A Brimble^{1

2

3}, Joseph J Gingell^{1

2}, Erica R Hendrikse¹, Annie Lovell¹, Nicole Prodan¹, Patrick M Sexton⁴, Andrew Siow^{1

3}, Christopher S Walker^{1

2}, Harriet A Watkins^{1

2}, Geoffrey M Williams^{2

3}, Denise Wootten⁴, Sung H Yang^{1

2}, Paul W R Harris^{1

2

3}, Debbie L Hay^{1

2}

Affiliations

¹ School of Biological Sciences, University of Auckland, Auckland, 1010, New Zealand.
² Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, 1010, New Zealand.
³ School of Chemical Sciences, University of Auckland, Auckland, 1010, New Zealand.
⁴ Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

Abstract

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP₁ production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM_15-52 G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.