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.