Our transcriptomic and genomic survey demonstrates that choanoflagellate GPCRomes are richer than previously understood, both in the number and diversity of GPCRs represented. We identified 18 distinct GPCR families in choanoflagellates, of which 12 – Rhodopsin, GPR155, GPR157, GPCR TKL/K, TMEM145, TMEM87, GPR180, Hi-GOLD, RSF, GPRch1, GPRch2, and GPRch3 – were newly described in thes organisms (Table 1, Figure 2).Among these families, we observed that aGPCRs generally constitute the largest class of GPCRs in choanoflagellates, apart from the GPCR TKL/K family in acanthoecids. most choanoflagellate GPCR families are conserved in metazoans or other eukaryotes.This supports the view that GPCRs are ancient gene families found across eukaryotic diversity (Krishnan et al., 2012; de Mendoza et al., 2014; Mojib and Kubanek, 2020).

In addition, by assessing the conservation of choanoflagellate GPCRs in other eukaryotes, we uncovered GPCR families in metazoans that, to our knowledge, had not been reported in this clade before. These include Hi-GOLD, GPRch3, and GPCR TKL/Ks. in addition, we found that five GPCR families – RSF, Hi-GOLD, GPRch3, GPR157, and GPRch1 – are conserved across diverse eukaryotes, thereby expanding the repertoire of GPCRs inferred at the root of the eukaryotic tree (de Mendoza et al., 2014). Future increases in the sequencing of non-metazoans will likely expand further the inferred diversity of GPCRs present in the progenitors of metazoans and other eukaryotes.

The identification of Glutamate Receptors, Rhodopsins, and aGPCRs in choanoflagellates, three GPCR families with relatively well-characterized subfamilies in metazoans (Fredriksson et al., 2003; Pin et al., 2003; Bjarnadóttir et al.,2004; Chun et al., 2012; Scholz et al., 2019; Ellaithy et al., 2020; Wittlake et al., 2021), led us to investigate whether these subfamilies are conserved in choanoflagellates and close relatives. Based on analyses of the 7TM domains,we found relatives of the main subfamilies of metazoan Glutamate Receptors in CRMs (Figure 3B). In contrast, most aGPCR subfamilies reported in metazoans have no obvious orthologs in CRMs, suggesting that metazoan and CRM aGPCRs diversified independently (Figure 5). One exception was the ADGRV family (Weston et al., 2004; Hamann et al., 2015; Kusuluri et al., 2021), for which clear orthologs are found in choanoflagellates and filastereans (this study and Krishnan et al., 2012; Peña et al., 2016).

Although Rhodopsins are the most abundant GPCRs in metazoans, we identified only three metazoan-like Rhodopsins in CRMs: two in choanoflagellates and one in an ichthyosporean. In addition, we uncovered Rhodopsin-l

Unraveling the Mystery of the HRM-GAIN-7TM Module in aGPCRs

Adhesion G protein-coupled receptors (aGPCRs) are a diverse family of transmembrane proteins crucial for cell signaling and adhesion in animals.Recent research has shed light on the evolutionary origins and structural characteristics of these receptors, notably focusing on a unique module: the HRM-GAIN-7TM. This module, comprised of a hormone Receptor Module (HRM), a GAIN domain, and seven transmembrane (7TM) helices, is central to understanding aGPCR function and evolution.

Historically,the HRM-GAIN-7TM module was believed to be exclusive to metazoans – animals. However, groundbreaking studies have revealed its presence in choanoflagellates and filastereans, which are among the closest living relatives to animals. This discovery pushes back the evolutionary origin of this module considerably, suggesting it arose in stem holozoans, the group encompassing animals and their unicellular relatives. The presence of this module in these organisms indicates it predates the evolution of complex multicellularity.

Researchers have observed a remarkable diversity in the N-terminal protein domain combinations within aGPCRs in choanoflagellates and filastereans. This diversification suggests an expansion of functional roles for these receptors even before the emergence of animals. The aGPCRs in these organisms exhibit all the key structural features of metazoan aGPCRs, including a long N-terminus containing a GAIN domain and multiple extracellular domains (ECDs). This structural conservation further supports the ancient origins of this receptor family.

The function of the HRM-GAIN-7TM module remains a significant puzzle.While it’s known to bind peptide hormones and neuroShima et al., 2004; Kimura et al., 2006; Araç et al., 2012; Prömel et al., 2012; Araç and Leon, 2019, the precise mechanisms and physiological roles are still under examination. Understanding this module is critical for deciphering the complex signaling pathways mediated by aGPCRs and their impact on advancement,immunity,and other essential biological processes.

Key Takeaways

• The HRM-GAIN-7TM module, previously thought to be exclusive to animals, is also found in choanoflagellates and filastereans.
• This discovery suggests the module evolved in stem holozoans, before the emergence of complex multicellularity.
• A diverse range of N-terminal domain combinations exists in aGPCRs of choanoflagellates and filastereans, hinting at functional diversification.
• The precise function of the HRM-GAIN-7TM module remains an area of active research.

Forward-Looking Statement: Ongoing research utilizing advanced structural biology techniques and functional assays promises to unlock the secrets of the HRM-GAIN-7TM module, providing valuable insights into the evolution of cell signaling and the development of novel therapeutic strategies.