6. The bigger picture – PAM from green algae to human
PAM and other granule membrane proteins are deposited on the cell surface during the process of exocytosis (Fig.4A ). Immature granules contain PAM and soluble cargo proteins. During the maturation process, cargo proteins are further modified; the granules move from the Golgi region towards the cell surface and acquire the cytosolic proteins needed to make them responsive to the right stimuli. Any PAM protein in the granule membrane appears on the cell surface as the soluble cargo proteins are released into extracellular space (Fig.4A , Exocytosis). Under normal conditions, PAM is rapidly removed from the plasma membrane (Fig.4A , Endocytosis) and returned to the secretory granules via a complex series of steps through the endocytic pathway. Clathrin coated vesicles mediate the removal of PAM from the cell surface and detailed studies of PAM trafficking in corticotrope tumor cells have tracked the internalized protein as it moves from early endosomes into late endosomes (Bäck et al., 2017; Rajagopal, Stone, Francone, Mains, & Eipper, 2009). Whether the PAM protein that exits late endosomes is degraded in lysosomes or is recycled to immature granules (Fig.4A ) is determined by O-linked sugars attached to the linker region between PHM and PAL, by a conserved pH-sensitive region that follows PHMcc and by the phosphorylation status of the PAM cytosolic domain (Rajagopal et al., 2009; Rao, Zavala, Deb Roy, Mains, & Eipper, 2019; K. Vishwanatha, Bäck, Mains, & Eipper, 2014; K. S. Vishwanatha, Bäck, Lam, Mains, & Eipper, 2016). Targeting its O-linked sugars or its pH-sensitive region could provide a means of preventing the return of PAM to the secretory pathway.
Compounds that stimulate the secretion of peptide hormones and neurotransmitters stored in granules increase the amount of PAM protein delivered to the plasma membrane (Fig.4A , lightning bolt). Tumor cells often secrete autocrine growth factors, many of which are amidated peptides (Jimenez et al., 2001). The appearance of PAM on the surface of these cells provides an opportunity for cell-impermeant PHM inhibitors or PAM-targeted tags that can be tracked in vivo to bind to its ectodomain and enter the endocytic pathway along with PAM. Internalization of a PHM inhibitor would be expected to reduce the synthesis of bioactive amidated growth factors while ectodomain antibodies or active-site targeted tags might facilitate tumor cell localization (Fig.4A ). An inactive prodrug might be expected to bind to PAM while it is exposed on the cell surface, with drug activation designed to occur in the lower pH environments encountered during endocytic trafficking. As discussed above, with an understanding of the structure of bifunctional PAM, the design of dual function drugs (targeting PHM and PAL) might provide a tool capable of preventing the endocytic trafficking needed to return PAM to the secretory pathway. Outside of its active sites, the sequences of insect and human PHM may be divergent enough to design species specific inhibitors useable as insecticides.
Phylogenetic studies have suggested several different situations in which controlling PAM activity or localization could be beneficial. While the active sites of C. reinhardtii PAM (CrPAM) include each of the residues identified as essential in vertebrate PHM and PAL, this unicellular green alga does not store peptides in secretory granules (Fig.4B ). CrPAM is localized to the membranes of the Golgi complex, as observed in vertebrates, but the presence of two prominent motile cilia made it clear that CrPAM is also localized to the ciliary membrane (Fig.4B ) (Kumar, Blaby-Haas, et al., 2016). Unlike the enzymes used by C. reinhardtii to respond to the need for various nutrients, neither PAM activity nor PAM protein is secreted through the classical secretory pathway (Kumar et al., 2017; Luxmi et al., 2019). In vertebrate systems, endoproteolytic cleavages that separate the PHM and PAL domains from the transmembrane domain result in their secretion under both basal and stimulated conditions (Kumar, Mains, et al., 2016). During vegetative growth, C. reinhardtii rely on bioactive ectosomes released from their cilia (Fig.4B , Inset) to destroy the mother cell wall (Wood, Huang, Diener, & Rosenbaum, 2013). Ciliary ectosomes also play a key role during sexual reproduction in C. reinhardtii (M. Cao et al., 2015). Active CrPAM is present in mating ectosomes, along with its amidated products, one of which acts as a chemomodulator (Luxmi et al., 2019) (Fig.4B ). PAM was subsequently identified in the ciliary membranes of vertebrate motile and sensory cilia (Kumar et al., 2017). The formation of ciliary ectosomes (Fig.4B ) is topologically similar to the formation of intraluminal vesicles in the endocytic pathway (Fig.4C ). Fusion of a multivesicular body containing ILVs results in their release as exosomes (Fig.4C ). PAM has been identified in intraluminal vesicles (Bäck et al., 2017) and in exosomes isolated from human saliva and urine (Gonzalez-Begne et al., 2009; Principe et al., 2013; Z. Wang, Hill, Luther, Hachey, & Schey, 2012). Much remains to be learned about the role played by ectosomes/exosomes, but the presence of PAM in these structures offers a means of isolating and characterizing that subset.
When PAM expression was reduced in C. reinhardtii , the cells were unable to form cilia (Fig.4D ) (Kumar et al., 2017). Instead of their normal pair of motile 12 µm long cilia (Fig.4B ), PAM knockdown cells had ciliary stubs containing microtubule fragments. The ciliary axoneme did not form and the appearance of the transition zone was altered. In humans, fully 5% of the genome is thought to be involved in the formation and function of cilia and ciliopathies are associated with many congenital diseases including obesity, blindness and kidney disease (Engle, Bansal, Antonellis, & Berbari, 2021; Leroy et al., 2021; Nager et al., 2017). The effects of reducing PAM levels on ciliogenesis are species specific, but occur in planaria, zebrafish and mouse (Fig.4D ). The hypothalamic neurons that produce proopiomelanocortin must have cilia and the melanocortin 4 receptor must be localized to those cilia in order to regulate appetite normally (Y. Wang et al., 2021). Much remains to be learned about the sensory and signaling roles of cilia and tools that allow the manipulation of ciliary PAM would be of great utility in these studies.
It is now known that the PAM protein has several non-catalytic functions (Fig.4E ). Regulated intramembrane proteolysis of PAM releases a soluble fragment of its cytosolic domain (sfCD). Nuclear accumulation of sfCD depends on its phosphorylation status and is thought to contribute to the ability of PAM to alter gene expression in a tissue-specific manner (Rajagopal et al., 2009). In atrial myocytes, which express PAM at levels higher than those observed in other cells, the PAM protein (whether active or inactive) plays an essential role in the formation of atrial granules, which store natriuretic peptides (Bäck, Luxmi, Powers, Mains, & Eipper, 2020). Instead of its catalytic activity, the ability of PAM to function as a re-usable luminal cargo receptor for proatrial natriuretic peptides early in the secretory pathway is thought to be essential to its ability to support granulogenesis.