there has been a lot of debate not only about how dcsg sorting occurs but also about exactly where in the cell this triage takes place .
all cells have the capacity to rapidly secrete proteins after their transit through the constitutive secretory pathway .
a great deal of evidence supports the view that in the appropriate cell type , dcsg sorting signals can redirect proteins from the constitutive secretory pathway to dcsgs , thus confirming that it is not a default secretory pathway .
some groups have proposed that dcsg sorting occurs through the action of a sorting  receptor  present in the tgn that latches onto granule - destined proteins at sites where nascent granules will bud ( chung et al . , 1989 ; cool et al . , 1997
this has been referred to as the  sorting by entry  model . on the other hand , convincing evidence has been presented that in cells that generate dcsgs , all of the contents of the tgn are initially encapsulated into the nascent granules ( arvan and castle , 1998 ) .
 model proposes that those proteins destined to be secreted constitutively are progressively extruded in low - density vesicles as the granule matures , ultimately leaving only the correct cargo protein in the mature dcsg .
the first truth is that , regardless of the site at which sorting occurs , a mechanism has to exist that establishes and then maintains the segregation of dcsg cargo proteins from those that are constitutively secreted .
thus , it is a reasonable postulate that some mechanism exists to anchor the appropriate cargo proteins to the dcsg as it forms or matures .
a second truth is that the sorting of proteins to dcsgs is a prerequisite for certain posttranslational processing steps in hormone and protease activation .
for example , the conversion of proinsulin to active insulin , the conversion of proopiomelanocortin ( pomc ) to its many peptides , including acth , and the proteolytic activation of prorenin to renin all occur only after the precursors are encapsulated in the nascent secretory granules ( orci et al . , 1986 ; taugner et al . , 1987
this makes sense for the organism because it ensures that the secretion of the active hormones or proteases is under appropriate physiological control .
however , for granule - restricted activation to occur , it is necessary that both the protein precursors and the appropriate processing enzymes end up in the same dcsg . in the case of proinsulin
, this means that the proprotein convertases pc1/3 and pc2 , as well as carboxypeptidase e ( cpe ) , all of which are required for generation of active insulin , have to be cotargeted with proinsulin in the budding granules .
thus , a second postulate is that a mechanism exists to ensure efficient cotargeting of protein precursors and their processing enzymes in the same organelle .
dcsgs also share , by definition , the distinguishing trait of a core that appears dark or dense in electron micrographs
. however , in spite of this common appearance , there may be important functional and mechanistic differences in dcsgs .
for example , the gonadotropes of the pituitary store luteinizing hormone and follicle - stimulating hormone in separate dcsgs , and their release is independently controlled ( for review see dannies , 2001 )
. likewise , there are two types of dcsg in chromaffin cells containing either epinephrine or norepinephrine , and these are morphologically distinct ( hendy et al . , 2006 ) . the signals for targeting proteins to dcsgs also show tissue - specific variations ; removal of 90 amino acids from the c terminus of the granin chromogranin a ( cga ) prevents its sorting to dcsgs in pituitary gh4 cells , but has no effect on dcsg sorting in sympathoadrenal pc12 cells ( cowley et al
. likewise , pomc is efficiently stored in dcsgs when transfected into cultured pituitary cells , but not in sympathetic neurons ( marx et al . , 1999 ) .
thus , a third truth is that not all dcsgs are alike , and it is a reasonable postulate that dcsgs can be assembled , even within the same cell , through more than one mechanism . could some of these truths explain the difficulties in reaching consensus on the protein signals necessary for dcsg targeting ?
there has been no shortage in the variety of dcsg sorting mechanisms proposed in the last 20 yr ; these include protein domains that interact with or that traverse membranes and that may or may not interact with additional proteins on the cytoplasmic side of the dcsg , proteins proposed to be a  master switch  for granule formation , universal granule cargo receptors , protein domains that mediate aggregation in the late tgn , certain paired basic protease cleavage sites or  helices in secretory proteins , disulfide - constrained loops , acidifying proton pumps , and other mechanisms . as a result , investigators have become progressively entrenched in defending their favorite mechanisms and commonly use the descriptors  controversial  and  difficult to repeat  to describe the work of others in their publications . nevertheless , it is possible to accommodate most of these findings in a model that subdivides targeting function into three components ( fig .
1 ) : membrane associated ( or traversing ) tethers , tether - associated cargo , and aggregation .
many dcsg cargo proteins also aggregate to form the dense core ( blue ) , and these aggregates may contain more than one protein .
the yellow boxes indicate the various protein domains or mechanisms that have been implicated in dcsg sorting .
note that some proteins ( such as insulin ) may have multiple dcsg sorting mechanisms .
peptidyl--amidating monooxygenase ( pam ) , phogrin , and muclin are all type 1 membrane - spanning proteins that are targeted to dcsgs ( bell - parikh et al . , 2001 ; wasmeier et al . , 2002
, the granule sorting domain is located in the cytoplasmic tail of the protein , and although the exact nature of the signal is still debated , it appears that this domain can bind the clathrin adaptor proteins ap-1 and -2 in vitro ( wasmeier et al .
such interactions might provide a means of communication between the granule cargo proteins and the membrane domains or cytoplasmic proteins that will define the budding dcsgs .
the membrane - binding domains of the granule - resident protein cpe ( cpe ; dhanvantari et al . , 2002 ) and the prohormone convertases pc1/3 ( jutras et al . , 2000 ; arnaoutova et al . , 2003 ) and pc2 ( assadi et al . , 2004 )
are also key for their targeting to dscgs , and there is agreement that the granule sorting is mediated by short  helical domains .
an  helical domain has also been shown to be important for targeting prosomatostatin ( mouchantaf et al . , 2001 ) ,
cga ( taupenot et al . , 2002 ) , and vgf ( garcia et al . , 2005 ) to dcsgs .
recent results suggest that  helices with the ability to direct granule sorting in secretory proteins share the characteristic of charge segregation from a hydrophobic patch , which is consistent with a shallow membrane interaction ( dikeakos et al . , 2007 ) .
this first group of dcsg proteins could therefore be said to be tethered to the membranes of the tgn or the maturing granule .
a second group of granule sorting domains may act by binding cargo proteins to these granule - tethered proteins .
for example , cpe has been proposed to interact with several granule cargo proteins including proenkephalin , proinsulin , pomc ( cool and loh , 1998 ) , brain - derived neurotropic factor ( probdnf ; lou et al . , 2005 ) , and
2005 ) to promote their retention in secretory granules , even though some of these are not enzymatic substrates of cpe .
no common mechanism for interaction of these cargo proteins with cpe has yet emerged , although undefined residues within an n - terminal disulfide - constrained hydrophobic loop in pomc ( loh et al . , 2002 ) and acidic residues in probdnf ( lou et al . , 2005 ) have been reported , and both seem to be important for sorting the respective proteins to dcsgs ( cool et al . , 1995 ; lou et al . , 2005 ) .
paired basic amino acids have also been reported to direct dcsg sorting in some proteins , including proneurotensin ( feliciangeli et al . , 2001 ) ,
prorenin ( brechler et al . , 1996 ) , prothyrotropin - releasing hormone ( mulcahy et al .
, 2005 ) , and progastrin ( bundgaard et al . , 2004 ) , and to increase the sorting efficiency of proinsulin ( kuliawat et al . , 2000 ) . in the analyses performed to date
, it appears that these paired basic amino acids must constitute a cleavage site for one of the granule - resident prohormone convertases ( pc1/3 or pc2 ) to function as a granule sorting domain because changing the cleavage site to one recognized by furin ( another member of the family that cleaves its substrates in the early secretory pathway ) causes the proteins to be secreted through the default constitutive pathway ( brechler et al . , 1996 ) .
these results raise the possibility that certain dcsg - targeted proteases can act as sorting chaperones for their substrates , in addition to being processing proteases .
muclin has also been suggested to act as a granule cargo receptor in pancreatic cells through its binding of sulfate groups on o - linked glycosylated proteins ( boulatnikov and de lisle , 2004 ) .
atrial natriuretic factor ( anf ) has also been shown to be tightly bound to the membranes of atrial myocyte secretory granules through its interaction with pam ( o'donnell et al . , 2003 ) , although it is not a substrate of pam .
thus , a variety of interactions with  tethers  may serve to target proteins to secretory granules . notably , if this mechanism is correct , it would , in some cases , provide a means to ensure that processing enzymes and their substrates end up in the same dcsgs .
a third category of granule - targeting mechanisms involves formation of high molecular weight protein complexes or aggregates .
indeed , many granule - targeted cargo proteins have the ability to multimerize or aggregate , leading , in most cases , to the formation of electron - dense cores .
a direct correlation between the ability to aggregate in vitro and to be sorted to secretory granules in transfected cells has been reported for rat pro - anf ( canaff et al . , 1996 ) and cga ( jain et al . , 2002 ) . because granins are acidic proteins that cluster in the slightly acidic environment present in dcsgs ( for review see dannies , 2001 )
it has been suggested that aggregation may serve to prevent their extrusion from the maturing granule .
( 2005 ) showed that treatment of pc12 cells with bafilomycin a1 , which is a specific inhibitor of vacuolar h - atpase , resulted in a decrease in regulated secretion of cga with a concomitant decrease in visible dcsgs , suggesting that regulated secretion of cga and dense core formation are linked in dcsgs .
kim et al . ( 2001 ) also showed that silencing cga expression in pc12 cells results in a loss of visible dcsgs , leading the authors to the striking conclusion that cga is not only a component of the dense core , but that it is also a  master regulator  of dcsg biogenesis .
( 2004 ) suggests that the role of cga in this process may not be that simple ; they found no correlation between dscg content and cga expression in isolated clonal lines of pc12 cells , suggesting that other proteins could be contributing to dcsg appearance .
in fact , expression of several other dcsg cargo proteins , including provasopressin , prooxytocin , pomc , secretogranin ii , and chromogranin b , is sufficient to induce aggregate - containing cytoplasmic vesicles , even in cells with no regulated secretory pathway ( beuret et al . , 2004 ) , although these probably do not display all of the functional characteristics of dcsgs ( meldolesi et al . , 2004 ) .
( 2004 ) recently reported that a polypyrimidine - binding protein ( ptb ) , which is up - regulated under conditions of high insulin demand , stabilizes messenger rnas of many of these same dcsg cargo proteins in insulin - producing cells and leads to increased granule formation .
cga has also been reported to induce the expression of pn-1 , which is a serine protease inhibitor that slows the turnover of several dcsg cargo proteins ( kim and loh , 2006 ) that could provide an additional mechanism for increasing dcsg aggregate formation . because cga binds to another granin partner , sgiii ( hosaka et al . , 2004 ) , which , in turn , can associate with cholesterol ( hosaka et al . , 2004 ) and cpe ( hosaka et al . , 2005 )
, aggregation may synergize with protein  protein and protein  membrane interactions to improve the retention of cargo proteins in the maturing granule and their regulated secretion .
in spite of the compelling arguments presented for these various dcsg sorting mechanisms , their translation to the whole animal has been anything but simple .
one example of this difficulty is the proposed role of cga as a master regulator of granule formation . although down - regulation of cga expression was reported to result in the loss of detectable dcsgs in cultured pc12 cells ( kim et al . , 2001 ) ,
( mahapatra et al . , 2005 ) or no discernable effect ( hendy et al . , 2006 ) on dcsg formation in the cga - rich adrenal chromaffin cells in two independent studies .
in spite of the differences in the effects on dcsg morphology , both groups report a similar and dramatic effect on catecholamine secretion in the cga - deficient mice , proving that cga deficiency is not entirely without consequence . how can these apparent differences in the requirement for cga be explained ?
one obvious possibility is that in vivo , other dcsg cargo proteins can complement the function of cga in the formation of the dense core , but can not compensate for its absence in catecholamine storage and secretion . in support of this possibility
, the group that saw no effect of cga inactivation on dcsg formation reported an up - regulation of cgb and sgii in the adrenal glands of the engineered mice ( hendy et al . , 2006 ) .
thus , although cga may affect dcsg formation in some cultured cells , this particular function can obviously be replaced in vivo . nevertheless ,
although experiments to date have not identified a master regulator of dcsg formation , the concept may not be entirely wrong in specific cell types ; anf inactivation in mice leads to a complete loss of visible dcsgs in the cardiac atrium ( john et al . ,
1995 ) , and inactivation of the renin gene leads to a complete disappearance of dcsgs in the juxtaglomerular cells of the kidney ( clark et al . ,
it 's important to note , however , that regulated secretion can occur in the absence of a dense core as it does in many neurosecretory vesicles . in the case of the anf and renin - deficient mice
, it will be intriguing to determine if the remaining cargo proteins are still packaged in such vesicles in the absence of the aggregating partner .
a similar conundrum exists with cpe as a sorting receptor for a variety of dcsg cargo proteins .
( 1997 ) originally proposed cpe as the regulated secretory pathway sorting receptor because they observed endocrine disorders in the cpe fat / fat mouse that harbors a mutation in the cpe gene .
proinsulin and pomc are among the several proteins that were shown to bind to cpe and that were proposed to enter dcsgs by this association ( cool and loh , 1998 ) .
however , recent results demonstrate that both proinsulin and pomc are correctly targeted to dcsgs in cpe fat / fat mice ( irminger et al . , 1997 ; hosaka et al . ,
although there may be many reasons why it has been hard to derive a consensus for the mechanisms and components of the dcsg sorting machinery , the most intuitive is that we are the victims of our own scientific reductionism , i.e. , that in our search for a simple canonical sorting mechanism we have developed a grossly oversimplified view of the way in which proteins enter dcsgs .
nearly 100% of the proinsulin produced in pancreatic  enters dcsgs ( rhodes and halban , 1987 ) , whereas only about 25% of the prorenin in the secretory pathway of kidney juxtaglomerular cells is sorted to dcsgs ( pratt et al . , 1987 ) .
proinsulin contains numerous potential dcsg sorting domains , such as a binding domain for cpe ( cool and loh , 1998 ) , two paired basic amino acid protease cleavage sites ( steiner et al . , 1996 ) , and the ability to hexamerize and subsequently aggregate ( quinn et al . , 1991 ) , whereas prorenin only contains a single dcsg sorting domain : a paired basic amino acid protease cleavage site ( brechler et al . , 1996 ) .
in the case of prorenin , changing even a single one of these basic amino acids completely eliminates dcsg targeting in tissue culture cells ( brechler et al . , 1996 ) .
in contrast , neither the mutation of the protease cleavage sites ( halban and irminger , 2003 ) nor the hexamerization domain ( quinn et al . , 1991 ) of proinsulin appears to affect its dcsg sorting .
combined with the finding that proinsulin is still efficiently sorted to dcsg in cpe - deficient mice ( irminger et al . , 1997 )
however , another possible explanation is that , with its many dcsg sorting signals , proinsulin might be able to compensate for the loss of any single sorting domain .
there is , in fact , some evidence to support the view that dcsg sorting signals can synergize ; duplicating the disulfide - constrained loop dcsg sorting signal normally found at the n terminus of cgb results in a greater sorting efficiency to dcsg than the native protein ( glombik et al . , 1999 ) .
furthermore , combination of  helical and paired basic amino acid sorting domains on either the same protein or on two proteins capable of dimerizing led to a dramatic increase in dcsg sorting over proteins containing either individual domain ( lacombe et al . , 2005 ) .
thus , diverse sorting signals may be able to functionally complement each other even through protein 
( 2005 ) also reported that pituitaries of the cpe fat / fat mouse contain elevated levels of both sgiii and cga that might compensate for the loss of cpe in targeting pomc to dcsgs .
all of these cases are consistent with the existence of multiple sorting mechanisms , each of which can contribute to the overall efficiency of protein sorting or retention in dcsgs .
cell types and the nature and/or the number of the sorting domains contained in the cargo protein would ultimately determine the extent to which each mechanism is active .
multimerization and aggregation could add synergy between mechanisms used by other dcsg cargo proteins in the aggregate .
with such a model , it 's also easy to imagine how changing conditions within the cell could alter dcsg sorting efficiency of a protein , which is a potential control point that has important implications for hormone secretion but has received little attention to date .
work to date has largely supported the three previously outlined postulates : ( a ) proteins exist that could explain the anchoring of dcsg cargo to the granule membrane , ( b ) protein complexes between processing enzymes and their substrates have been proposed that could explain how these end up in the same granules , and ( c ) the diversity of sorting mechanisms helps to understand how there could be distinct dcsgs even within single cells and how there could be such a lack of agreement on the mechanism of dcsg sorting .
unfortunately , this lack of simple consensus has limited progress , primarily because of attempts to determine the role of single targeting motifs in given proteins in various cell culture models that may or may not be entirely appropriate . although this approach has certainly not been without worth , a better understanding of the complexity of this important cellular event may help to design experiments that will help to significantly advance this field of research .
how does the dcsg protein cargo identify the membrane patches that will make up the mature granule ?
how do these complexes communicate with the cytoplasmic accessory proteins that are necessary for the formation of the budding dcsgs ?
in fact , although it has been difficult to explain the entry of proteins into dcsgs , we can expect that describing the assembly of proteins on the surface of the dcsgs that are necessary for their cytoplasmic transport , docking at the membrane , and exocytosis will be an equally daunting challenge . a recent report on the components of the functionally related synaptic vesicle identified > 400 associated proteins ( takamori et al . , 2006 ) .
what is abundantly clear is that in the characterization of this unique organelle , there is still a lot to sort out .
 the intracellular sorting of peptide hormone precursors to the dense core secretory granules ( dcsgs ) is essential for their bioactivation . despite the fundamental importance of this cellular process 
 , the nature of the sorting signals for entry of proteins into dcsgs remains a source of vigorous debate . 
 this review highlights recent discoveries that are consistent with a model in which several protein domains , acting in a cell - specific fashion and at different steps in the sorting process , act in concert to regulate the entry of proteins into dcsgs . 
Three truths and three postulates
A plethora of signals, a paucity of consensus
In 
Synergy and diversity in granule sorting mechanisms
Conclusion