Novel Genetic Variant of a Class B G Protein-Coupled Receptor Reveals a Role for Adrenomedullin in Human Fertility

The adrenomedullin (AM) peptide receptor system has been proposed as a key regulator of lymphatic/blood vascular development from animal models for almost two decades, with more recent data linking this to fertility.¹⁻⁸ A recent report identifies an in-frame deletion in an AM receptor subunit, CALCRL, in six members of a consanguineous family that causes a loss-of-function phenotype and likely explains multiple miscarriages.⁹ This important study provides vital data to support a similar role for this peptide-receptor system in humans. Mackie and colleagues conducted an elegant series of translational studies, whereby they discovered a novel recessive form of generalized lymphatic dysplasia that exhibits as nonimmune hyrops fetalis with fatal demise or clinical subfertility, in the homozygous or heterozygous states, respectively. They went on to characterize the underlying genetic defect and develop novel transgenic mice to link genotype to phenotype.

The calcitonin-like receptor (CALCRL; protein abbreviation CLR¹⁰) is a class B G protein-coupled receptor. This receptor is necessary for AM signaling and this gene is necessary for survival in mice (Calcrl).³ An accessory protein, receptor activity-modifying protein 2 (RAMP2) chaperones CLR to the cell surface and forms an integral part of the AM binding site.^11,12 Together, these proteins form the AM₁ receptor.¹⁰Ramp2 transgenic mice mirror Adm and Calcrl transgenic mice in many ways,¹ supporting the concept that together these proteins form a functional system for AM signaling. Given the conservation of the AM receptor system in other mammals, including humans, these alluring mouse studies have implied that AM may be responsible for controlling lymphatic vascular function and fertility in other species. However, direct evidence has been elusive, until now.

Mackie and colleagues identified a genetic variant of CALCRL in the family. This is an in-frame deletion of Valine 205, which is found within the first extracellular loop (ECL1) of CLR (Figure ​Figure11). In prior mutagenesis studies this amino acid had been substituted to alanine with unremarkable effect—a small reduction in AM potency and no effect on cell surface expression. Therefore, the suggested drastic functional consequences of the deletion may seem surprising. However, alteration of an amino acid side chain is much more subtle than a deletion, which has the potential to substantially affect protein structure, as was predicted by the molecular modeling presented in the paper. The modeled defect in the ECL1 structure was accompanied by functional data showing that the V205 deletion receptor had the impaired ability to traffic RAMP2 to the cell surface because the proteins had a diminished ability to interact. Functionally, the AM receptor containing the CLR mutant was still able to be activated by AM but at approximately 10-fold lower potency than the wild-type receptor. It is possible that there was substantial receptor reserve in the system, and that, in an individual expressing the receptor variant, receptor signaling could be substantially lower. On the other hand, it is known that AM haploinsufficiency has significant impact in mice,⁴ and thus the 10-fold reduction in potency could well be sufficient to yield pathophysiological outcomes. Additional mouse models supported the argument that haploinsufficiancy in different elements of the AM receptor system could indeed yield outcomes in line with the human phenotype, and this led to the conclusion that the CALCRL variant produced the clinical phenotpye. A transgenic mouse with the V205 deletion would be incredibly interesting but this would not be trivial to develop.

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Figure 1

Cryo-EM structure of the CLR/RAMP1 CGRP receptor with CGRP bound,¹⁶ showing the overall receptor structure including G protein (A), with the ECL1 region circled with the dashed line. (B) Closer view of ECL1 in this receptor with V205 colored red. No full length CLR/RAMP2 receptor structure is available, but it is expected that ECL1 would be similar.

This is a highly significant study because many studies of genetic variants of receptors do not adequately investigate the pharmacological and physiological consequences on receptor function. The study provides much needed answers to a family who have been plagued with fertility issues. This important finding should spur on studies relating to the AM system in fertility and further drug discovery efforts toward this receptor family. It is too early to say whether defects in AM signaling underlie other human pathologies but there is evidence for altered AM signaling in endometriosis, preclampsia, and septic shock.¹³⁻¹⁵ Given that CLR also forms a calcitonin gene-related peptide receptor together with RAMP1, there is also scope for genetic variants in CLR to contribute to other conditions.

Notes

This article is made available for a limited time sponsored by ACS under the ACS Free to Read License, which permits copying and redistribution of the article for non-commercial scholarly purposes.

Notes

The author declares no competing financial interest.

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