G protein-coupled receptors (GPCRs) are transmembrane proteins that modulate physiology across diverse tissues in response to extracellular signals. GPCR signalling can differ due to variation in the sequence (e.g. polymorphisms) or in the expression of receptors in different tissues. The resulting differences in response are an important source of physiological signalling bias. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms that can have distinct patterns of expression in human tissues. Here, we report the findings from a comprehensive study, integrating data from human tissue-level transcriptomes, GPCR sequences and structures, functional annotations, proteomics, single-cell RNA sequencing, population-wide genetic association studies, and pharmacological experiments. Our results show how a single GPCR gene can diversify into multiple isoforms with distinct structural and signalling properties, and how unique combinations of these isoforms can be expressed in different human tissues, contributing to differences in physiological signalling. Based on their structural changes and expression patterns, some of the detected isoforms may also influence drug response and represent new drug targets with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling, in which one considers how the combinatorial expression of receptor isoforms in a specific system (i.e. a particular cell type, tissue, or organism) collectively impacts receptor signalling. These observations pave the way for understanding the impact of isoform variation on GPCR signalling response and have implications for exploiting such variation as a source of GPCR selectivity in drug development.

Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of β-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of β-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting β-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is β-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of β-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.

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IT is well known that different organisms have the property of oxidizing iodides to free iodine. This reaction was first described by Sch6nbein [1848]. It was used by Bach & Chodat [1904] and among other workers by Wolff [1914; 1917] who suggested that the oxidation was due to a secondary reaction between the iodide and the oxidation products of polyphenols. Raciborski [1905], however, found that A8pergillus niger excreted an iodide-oxidizing system into its medium, but that both phenol oxidases and peroxidase were absent. Hence he postulated the existence of a specific iodide ooxidase. Korczewski [1922] confirmed Ra"ciborski's observation and showed that the iodide-oxidizing system was destroyed by heat and acid and inhibited by KCN, NH20H and peroxide. Further, if the iodine is not removed an equilibrium is reached and the reaction soon comes to an end. He always found peroxide present in the medium, but, since its concentration was very low, he concluded that it could not be responsible for the oxidation of iodide. He found, however, that the addition of peroxidase caused a marked acoeleration of iodide oxidation. Korczewski concluded that the culture medium contains an oxygenase similar to that postulated by Bach & Chodat [1902; 1903;' 1904], and that this enzyme forms with molecular oxygen a superoxide which is responsible for the oxidation of iodide. Methods The methods used for preparing an active iodide-oxidizing medium were basedon Korczewski's work. A.' niger was grown on a medium composed of: 4 % sucrose, 0-2 % K2HPO4, 1 %°(NH4)2S04, 0 05 % MgSO4, 7H20. It was found