345
ClinicalScience (1986)71,345-355
EDITORIAL REVIEW
New possibilities for intracellular renin and inactive renin now that
the structure of the human renin gene has been elucidated
BRIAN J. MORRIS
Molecular Biology and Hypertension Laboratory, Department of Physiology, The University of Sydney, New South
Wales, Australia
The recent determination of the structure of the
human renin gene [l-71 (described most completely
in [4])provides a wealth of basic essential information from which much insight can be gained concerning aspects of the biochemical nature and
function of the renin system. In the first part of this
review I will show how the new gene data can be
interpreted to explain the retention of renin by the
cell, which is important in the light of new findings
(also reviewed) regarding a renin-angiotensin
system in extrarenal tissue and the existence of an
intracellular system operating independently of the
traditional one in the bloodstream. The second
major theme of this review will concern inactive
renin, where I will draw on sequence data for prorenin derived from coding sequences in the gene
and other data to establish the basic biochemical
nature of inactive renin, and in so doing reconcile
seemingly contradictory reports in the literature on
this subject, particularly regarding its molecular
weight and activation.
Several potential renin precursors
To start with, the gene has not one, but three potential promoter elements ('TATA' sequences) where
RNA polymerase might bind to yield as many as
four different in-frame renin precursors (Figs. 1,2).
Promoter 1 is clearly the one used predominantly in
the human kidney (based on cDNA cloned from
human renin mRNA [8]) and yields a renin precursor with a hydrophobic N-terminal ('signal') peptide
that destines the processed product for eventual
secretion from the juxtaglomerular cell, by assisting
its entry into the cisterna of the endoplasmic reticulum. (The equivalent promoter is also the major one
used in the kidney and submandibular gland of the
Correspondence: Dr B. J. Morns, Molecular Biology
and Hypertension Laboratory (Building Fl3), Department of Physiology, The University of Sydney, NSW
2006, Australia.
mouse, based on biosynthetic studies [9-121 and
analysis of mRNA transcripts [ 13,141.)
There are, however, quite different possibilities.
These might operate to a greater or lesser extent in
the kidney and extrarenal tissues. Promoter 2,
located 48 base-pairs upstream from promoter 1, is
used in the mouse [13, 141 and has the potential to
yield a renin precursor with 27 extra amino acids
(Figs. 1,2). Putative promoter 3, located within the
first intron, if used, might yield either a renin precursor that lacks 48 amino acids [6], i.e. missing the
23 amino acid signal peptide and the N-terminal
(inhibitory [15])half of the pro region, or a precursor with a hydrophobic, signal-peptide-like leader
sequence, the 23 end amino acids of which could,
potentially, be removed by cleavage after a dibasic
(Lys-Arg) pair to yield the former (Figs. 1, 2). The
extended precursor arising from promoter 2 and
the shorter of the precursors from promoter 3
would each have a hydrophilic N-terminal leader
sequence and would therefore not enter the secretory compartment of the cell. Thus they may give
renin(s) that are locked within the cell to serve intracellular function(s).
Another way in which different proteins may
arise from the same gene is by differential splicing.
This is known to occur for several gene transcripts
(e.g., those for growth hormone, prolactin, insulinlike growth factor II, calcitonin, gastrin-releasing
peptide, nerve growth factor and high and low M,
kininogens) and involves alternative processing
mechanisms in the cutting and splicing of primary
mRNA transcripts in the nucleus, by which introns
are normally removed. There is, however, no evidence yet for differential splicing of primary mRNA
transcripts from the renin gene.
The renin gene is expressed in several tissues
The renin gene is present in all cells, but only certain cells have the necessary ancillary mechanisms
346
B. J. Morris
FIG.1. Sequence of the human renin gene and of preprorenin which is encoded on 10 exons interrupted by
long stretches of seemingly ‘nonsense’ DNA (introns).The amino acids of renin are numbered 1-340 above
the sequence; those of the ‘prepro’ (precursor) region are numbered -66 to - 1. Processing sites for
removal of signal peptide ( - 66 to - 44) and activation peptide ( - 43 to - 1) are indicated by large open
arrows. The nucleotides in the 5’ flanking DNA are numbered beneath the sequence in a negative direction
beginning with the likely major transcription start site (large solid arrow). Three potential promoters (‘TATA
boxes’) can be discerned (1, 2 and 3) where RNA polymerase might bind and begin transcription approximately 30 nucleotides downstream of each (solid arrows). Promoter 1 yields preprorenin (underlined).
‘Promoter 2’ would yield a precursor with 27 additional amino acids (italicized).‘Promoter 3’ (located within
New ideas from the renin gene
347
intron 1)would yield precursor(s)lacking the amino acids encoded in exon 1and having either a new hydrophobic region of 18 amino acids (italicized)or also lacking the first five amino acids in exon 2. The Nterminal methionine in each case is indicated ( 0 ) .Promoter 1 is preceded by a region of perfect dyad
symmetry (.**-*)and a ‘CAM box’ (underlined);less perfect dyad symmetry precedes promoter 2. Consensus sequences for binding of the glucocorticoid-receptor complex are indicated (- - - - -). Likely glycosylation
sites are shown, together with active-site aspartic acid residues (open stars).Residues in renin that lie within
the specificitypockets of the active site cleft and that are < 0.45 nm from substrate residues surrounding the
scissile bond are shown as S,-S,’. The flap across the active site involves amino acids 79-91.
B. J. Morris
348
GENE
Putative promoters
-
3
3 1
Ikb,
PROTEIN PRODUCTS
Promoter
used
1.
PRE
PRO
RENIN
45 k D a
( Major renal 1
\
Hydrophobic leader (signal peptide
\
\
)
+ Enters secretory apparatus
(Export from cell 1
43 k D a
3.
40 k D a
( R e n i n acts i n s i d e cell?
2f-
2. ?
48 k D a
FIG.2. The human renin gene has the potential to produce several different renin precursors. Depending on the promoter used the renin product may serve different functions.
Available evidence suggests that promoter 1 is the major promoter used in the kidney;
FWA polymerase would attach to it and transcribe a mRNA encoding preprorenin of
45 023 daltons. The alternative promoters might be used to a greater or lesser extent in
the kidney and extrarenal tissues. Use of ‘promoter 2’ could yield a 48 084 daltons precursor; as this has a hydrophilic N-terminal sequence it may not enter the secretory compartment of the cell. The use of ‘promoter 3’ could give either a 42844 daltons or a
39 811 daltons precursor having, respectively, hydrophobic and hydrophilic N-termini
which may determine whether or not the nascent polypeptide enters the secretory compartment. All of these molecular sizes refer to the unglycosylated translation product; it
should be noted, of course, that signal peptides are probably cleaved off in the cisterna of
the endoplasmic reticulum before completion of synthesis of the nascent chain. Potential
cleavage sites are shown as small arrows.
which elicit its transcription. In all species renin
gene expression is highest in the juxtaglomerular
cells of the kidney [16], consistent with the classical
view of renin as a major regulator of cardiovascular
homoeostasis and salt balance via enzymatic
generation of angiotensin I (ANG I) in plasma after
secretion appropriate to the circumstances. Other
tissues, however, also contain renin. The specific
cellular concentration, though, is lower. These
tissues include human chorion [17, 181, adrenal
[19-211, testicular Leydig cells [21, 221, pituitary
LH-gonadotrops and somatotrops [21,23-271, thyroid [21], aorta [28], brain [29, 301 and neuroblastoma x glioma cell lines [30, 311. Synthesis
occurs in submandibular glands, but is speciesspecific [32];although very high in the whole glands
of mice, it is similar to or less than kidney on a per
cell basis [16], as only 0.01% of kidney cells (in
man) synthesize renin (calculated from [33]).
Renin gene expression in testis, thyroid and
adrenal might be due to the local high concentrations of the specific hormones produced in these
tissues, as (dihydr0)testosterone and thyroxine can
directly enhance renin gene expression in mouse
submandibular gland [2, 3, 5, 161 and consensus
sequences for the binding of glucocorticoid-receptor complexes have been discovered in the
upstream regulatory region of the human renin gene
[4, 51 (Fig. 1).In support of this, it is noteworthy
that: (i) administration of corticotropin to hypophysectomized rats (which would increase adrenal
corticosteroid production) is accompanied by a 40fold increase in adrenal renin [20], (ii) adrenal renin
is increased in Cushing’s disease caused by adrenal
cortical hyperplasia, and decreased in aldosteronoma tissue [191, and (iii) both hypophysectomy
and oestrogen decrease testicular renin [22]. Since
ANG II stimulates DNA synthesis and growth of
New ideas from the renin gene
adrenal cortical cells [34], a local adrenal, reninangiotensin system could help corticotropin maintain adrenal weight.
Extrarenal renin may represent uptake of renal
renin carried in the bloodstream or expression of
the renin gene in extrarenal tissues. Although recent
experimental evidence indicates that most or all of
the renin in walls of blood vessels originates from
specific sequestration of plasma renin supplied
from renal secretion [35-371, the detection of reninlike mFWA in chorion [38], adrenal [39] and testis
[39] strongly supports renin gene expression and
synthesis of renin at several extrarenal sites. Renin
in regional vascular beds generates SO-90% of total
plasma ANG I [37] by its highly specific hydrolysis
of the only known natural substrate, angiotensinogen, supplied to the bloodstream by the liver; ANG
I is then hydrolysed to ANG I1 by a ubiquitous
dipeptidyl ecto-peptidase (converting enzyme) and
ANG I1 acts on specific receptors in the vicinity to
maintain peripheral vascular resistance [37]. Other
(non-vascular) renin-containing cells have been
reported to contain all components of the system.
These include neuroblastoma [30, 311, phaeochromocytoma [19], adrenal cortical [40] and Leydig [40] cells. As well, cloned juxtaglomerular cells
have been reported to contain every component
[41]. Moreover, there is now good evidence for
extrahepatic synthesis of angiotensinogen, namely
the recent detection of angiotensinogen mRNA in
extracts of brain [42] and kidney [43, 441 and in
granular duct cells of mouse submandibular gland
349
granules [60], which contain material ingested from
extracellular fluid, it has been suggested that intragranular ANG I1 has an extracellular origin [59].
ANG I can be detected, though, after treatment
with captopril[61] and has been found, recently, in
granules [62]. Furthermore, the kidney contains
angiotensinogen in granules [63] and angiotensinogen mRNA has now been detected as well [43].
Thus the possibility of an intracellular system in
kidney [63, 641 remains. In addition, ANG I has
been demonstrated recently in the uterus, salivary
gland, testis and epididymis of man [65].
ANG II may act, as well, within the cell. For
example, ANG II becomes localized in the nucleus
of rat cardiac cells [66] and can stimulate myocardial mRNA and protein synthesis [67]. Moreover, chromatin (at least in liver and thymus)
contains a specific, high-affinity receptor for ANG
I1 [68].Interaction of ANG I1 with this protein leads
to unwinding of DNA, suggesting a role in enhancing transcription of specific genes. Indeed an ‘intracrine’ function has now been suggested for ANG II
[69], in addition to known endocrine, paracrine and
neurotransmitter functions.
Although there is potential for intracellular renin
to be derived from precursor( s) that involve hydrophilic leader sequences, at least a portion of the
renin in the pituitary and adrenal glands is likely to
be synthesized by way of hydrophobic leader (signal
peptide) sequence(s), as cell fractionation has
localized renin within granules in these tissues [19,
251.
WI.
Intracellular renin-angiotensin system
The molecular nature of inactive renin is now
clearer
It is noteworthy that actual secretion of renin
appears restricted to the juxtaglomerular cells and
chorion, so that most of the extrarenal renin may
remain locked inside the cells. Any secretory product may, in fact, be ANG 11. This may then subserve local (autocrine) functions depending on the
site, e.g. local regulation of vascular tone [37, 451,
stimulation of aldosterone [20] and adrenal growth
[34] and control of pituitary hormone secretion
[46-501. In the central nervous system ANG 11may
function as a neurotransmitter [49,5 11.
An intracellular renin may generate angiotensin
within cells. (Such a reaction need not necessarily
be limited to renin, however, as cathepsin D [52]
and other unidentified aspartyl proteases in plasma
[53] and brain 1541 can produce ANG I; furthermore, ANG 11 can be produced directly by tonin
[55], trypsin [56], kallikrein [56] and cathepsin G
[57].) Both renin and ANG 11 are present together
in the secretory granules of the juxtaglomerular cells
[58, 591. Because of the lysosomal nature of the
The second aspect of this review relates to ‘inactive
renin’, which, when discovered in 1970, Morris and
Lumbers suggested was a precursor or proteinbound form of renin [70, 711. This form has low
activity, is bigger than renin and can be activated by
proteases.
The new gene data provide at last concrete
molecular information about renin and precursor
polypeptides. Use of the major promoter in the
renin gene would lead to synthesis of the 406 amino
acid polypeptide underlined in Fig. 1. Signal (‘pre’)
peptide and activation (‘pro’) peptide are removed
by hydrolysis at the sites shown, as recently
established by biosynthetic studies in vitro [72] and
by partial amino acid sequencing of the N-terminus
of human renal renin (W. A. Hseuh, Y. S. Do & T.
Shinagawa, personal communication). Thus the
human renin polypeptide has a molecular mass of
37 200 daltons. The 43 amino acid activation (‘pro’)
peptide is 5105 daltons. In addition, human renin
contains oligosaccharide moieties which contribute
350
B. J. Morris
to its overall molecular weight. These are likely to
be attached at AsnS and
by N-glycosidic
linkage [2-81 (Fig. 1).In hog renin carbohydrate
comprises -3% of the molecule [73]. Moreover,
the elution from concanavalin A-Sepharose of
rabbit renin and half of crude human renal renin
with 5 mmol/l a-methyl mannoside suggests at least
half of the renin in man has a carbohydrate component of similar magnitude. Furthermore, recent biosynthetic studies [72] show that glycosylated and
unglycosylated prorenin differ in MI on SDS gels by
4000. Carbohydrate residues are close to a
hydrophobic surface, which enhances the interaction of renin with concanavalin A [74].Examination
of the tertiary structure [75] suggests that this surface may be near
(A second, more strongly
binding renin peak requiring 500 mmol/l mannoside [74] or 1 mol/l NaCl [76] for elution suggests
an additional component, or interaction, for half of
human renin, and could be relevant to the discussion later.)
Thus the molecular weight of the human renin
glycoprotein would be 39 000-4 1000, which
agrees well with estimates for the pure protein on
SDS gels [77, 781. The molecular weight of the activation (‘pro’) peptide (5105), when added to this
value, gives a molecular weight for prorenin of
44 000-46 000. This is consistent with values first
reported by my colleagues and I: (i) for prorenin in
rat kidney (in experiments involving isolated renin
storage granules rather than kidney extracts, so protecting prorenin from high renal protease activity,
binding proteins and extragranular modifications)
[64], (ii) for half of the inactive renin in normal
human plasma [76]and (iii)in incorporation experiments in vitro in mice, where synthesis of prorenin
was shown for the first time [9-121, and allowing for
the absence of glycosylation of renin in these experiments. Moreover, a prorenin-like polypeptide
purified recently from human kidneys [78]ran at MI
48 000 on SDS gels and at M, 5 1000 on Sephadex
G-100. In this context, it is worth pointing out that
the fact that more carbohydrate is exposed after
activation [79] argues against assertions that the
high apparent molecular weight of ‘inactive renin’ is
due merely to interactions of oligosaccharide with
the gel support matrix.
As calculated above, if ‘inactive renin’ is prorenin
it should have a molecular weight in the vicinity of
44 000-46 000. However, human plasma and
kidney extracts contain not only a MI
46 000-5 1000 form of ‘inactive renin’ (consistent
with the size of prorenin), but also equal quantities
of a MI 5 5 000-62 000 form [76, 801 that has attributes in common with prorenin, but is much bigger
and has partial activity. Both prorenin and this ‘big
inactive renin’ can be activated by acid or protease.
-
Moreover, big inactive renin is the major form seen
in renin-secreting tumours [ 8 11, diabetic nephropathy, nephrotic syndrome, pregnancy [76],
chorion cells and amniotic fluid [82].
What is this ‘big inactive r e d ? Since there is
only one gene in man [4] it must originate from the
same DNA coding sequence (Fig. 1) on chromosome 1 [83]. But could it represent the product of
an alternative transcript of the gene? Several
laboratories have published results of cell-free
translation experiments in which, as well as the
major product ( M , 45000), there are two minor
products, one of MI 55 000-60 000 and the other of
M , 40 000-43 000 (on SDS-electrophoretic gels) in
man [84] and mouse [ l l , 12, 851. (The smaller of
these may represent degradation of MI 45 023 preprorenin arising from the major promoter, 1. Alternatively, it could reflect one of the renin
precursor(s) (of M, 42844 and M , 39811 in Fig. 2)
that might arise from use of ‘promoter 3’.) Use of
‘promoter 2’ could yield a (nonglycosylated) renin
precursor polypeptide of 48 084 daltons. However,
such a protein has not been detected on SDS gels
after cell-free translation of renin mRNA from
human kidney [84]. A minor band does, however,
appear at MI 56000 [84]. Interestingly, if such a
large precursor were synthesized in cells it would
have the potential for additional glycosylation, at its
fourteenth amino acid (i.e. at Am*-Leu-Ser in Fig.
l ) , and this would contribute to a further elevation
in apparent M,. The low quantities, however, argue
against this being an explanation for ‘big inactive
renin’.
In looking for the nature of this ‘big inactive
renin’ it would be nice to reconcile many observations in the literature. Although human r e i n runs
at M, -40000 on SDS gels, its MI has been
reported to be 50 000 if purification is performed
under protease-free conditions [77]. This size is
compatible with the size of the putative precursor
polypeptide arising from ‘promoter 2’, as described
above, and that of the renin precursor synthesized
in human juxtaglomerular cell tumours [8l], but is
not consistent with the size of the human renal renin
precursor predicted from molecular cloning [8],
cell-free translation [84] and other experiments.
Thus big inactive renin secreted into plasma is
probably not a product arising from use of an
alternative promoter.
In seeking the answer, let us then examine what
happens after synthesis. Inactive (pro)renin of M,
44 000 and 48 000 can be found in renin granules in
rat [64] and man [86], respectively. Activation most
likely occurs within the juxtaglomerular cell, but
could occur later. For example, by kallikrein in the
richly granulated renal peripolar cells located in the
origin of the glomerular tuft [87] and in this way
New ideasfrom the renin gene
could contribute to the regulation of glomerular filtration via the high-affinity ANG I1 receptors on
nearby mesangial cells [88].
After secretion renin enters the circulation from
renal interstitial fluid [89]. Subsequently, bloodborne renin is filtered [90] and taken up by reabsorptive pinocytosis in the proximal tubule [90,
911, which contains a specific renin-binding protein
[92-951, and is then catabolized [91]. Could this
binding protein, then, explain 'big inactive renin'?
Human renin-binding protein is a glycoprotein of
apparent M, 43000 [95]. Its renal concentration
exceeds that of renin [92, 951 and binding inhibits
renin 80% [95]. An endogenous renal (thiol-)protease normally dissociates renin from the complex
[93],as does acidification.There is also evidence for
dissociation of the complex during gel filtration
[92],which would account for estimates of only M,
54000-62000 for 'big inactive renin', if it were
such a renin-protein complex. Renin-binding protein probably functions in the metabolic clearance
of renin. (The possibility of local actions of renin in
the proximal tubule, however, cannot be excluded,
especially as ANG 11regulates sodium reabsorption
by a direct action on proximal tubule cells [96],
which might contain at least some of the angiotensinogen granules in kidney [63].)The fact, however, that there is no evidence for renin-binding
protein in plasma [97] argues against it being
responsible for 'big inactive renin' other than in
extracts of kidney. In plasma, kinetics favour formation of a dissociable complex of renin, or an activation intermediate,with angiotensinogen.
Recently, M, 55000-62000, as well as M,
46 000-47 000 and M, 40 000-43 000 forms, have
been detected on SDS gels with antirenin, when
kidney extracts were prepared rapidly (W. A.
Hseuh, Y. S. Do & T. Shinagawa, personal communication). Thus 'big inactive renin' behaves as if
it were a single polypeptide unit. Furthermore, all of
the inactive renin in human plasma and kidney has
been found recently to be absorbed by antk'profragment' antibodies, suggesting strongly that it is
related to prorenin [98, 991. In the case of plasma
inactive renin, when antibodies were prepared from
synthetic peptides corresponding to different sections of the profragment sequence (predicted from
the genetic coding sequence), the only antibody to
bind was that directed against the C-terminal portion of the propeptide [72]. Thus plasma inactive
renin may have lost at least 32 of the 43 amino
acids in its profragment and be a truncated, activation intermediate in the conversion of prorenin to
renin [72]. This would account for the partial
activity of big inactive renin in biological fluids, the
capacity for further increases in activity, and the
fact that there is little detectable change in size after
35 1
activation. Alternatively, the N-terminal of the
profragment may be buried, as is the case with pepsinogen, and therefore protected from antibody. As
might be expected, the binding of these antibodies
shields the activation site from activation by trypsin
[72,99].
As alluded to above, the size of the activated
inactive renin in human plasma [76], amniotic fluid
[82] and juxtaglomerular tumour tissue [81] is considerably bigger ( M , 55 000-62 000) than the active
form seen in kidney extracts or -after cell-free
synthesis in vitro ( M , 40 000-43 000) [72]. Moreover, 'big inactive renin' in plasma in one study was
found to undergo a reduction in M, to 48 000 with
trypsin-activation and then to M, 43000 active
renin with glandular kallikrein (although with a pl
lower than that of active renin) [loo], indicating
substantial losses of polypeptide, the nature of
which would appear to be unrelated to renin or a
truncated profragment. All of this, together with
findings (i) of active M, 50000 human renal renin
(on electrophoretic gels) that is reduced to M,
39000 if protease persists in the extracts [77], (ii)
that activation of M, 55 000-62 000 'inactive renin'
results in an almost undetectable decrease in size
[76,80-821, (iii) of only one major cell-free translation product of M, 45000 for human renal renin
mRNA [72,84] and (iv)of the synthesis in vitro of a
M, 55 000 precursor in cultures of juxtaglomerular
tumour cells [81], implies that 'big inactive renin'
might be a partially active, truncated prorenin with
an additional component attached to the renin portion co- or post-translationally. This could have
something to do with the strongly binding fraction
on concanavalin A [74, 761, discussed above (and
which would thus contain glucose, mannose and
fructose, possibly added by 0-, as well as Nglycosylation). Its apparently large size ( M,
lOOOO), however, and the ability of protease
activity to remove it, is consistent with it comprising
polypeptide as well. Whatever this component is it
would appear that it is tightly (covalently?)attached
to the renin precursor.
The attached component could serve as a signal
for uptake of prorenin by the heart or blood vessels.
Interestingly, the very high concentrations of big
inactive renin in plasma in the first trimester of
pregnancy [76] coincide with maternal cardiac
enlargement and, coupled with findings of stimulation of protein synthesis by ANG II in the heart [66,
671, may point to a physiological role for big inactive renin, whose concentration, even in normal
individuals, exceeds that of renin by over three-fold.
Indeed, the major site of formation of ANG 11, the
pulmonary vascular bed, may also be a site of
sequestration of circulating big inactive renin; ANG
II would then be supplied immediately to the heart
-
352
B. J. Morris
downstream. In rats, big inactive renin has been
found to increase in plasma after nephrectomy
[loll in a manner that suggests mobilization from
cardiovascular stores. Furthermore, it may be that
loss of the major source (the kidney) could lead to
switching on of renin gene expression in extrarenal
vascular beds to maintain local needs. Expression
may also be induced when vascular cells are cultured in vitro.
Multiple pathways for renin synthesis
Two pathways have now been postulated for the
synthesis of human renal renix the conventional
route via the secretory compartment, that allows
activation of prorenin, and a cytoplasmic route, that
does not result in processing of precursor to renin
[81]. This concept is supported by recent immunocytochemical evidence [102]: as well as the classical
gradient of staining through the rough endoplasmic
reticulum, Golgi complex, protogranules and
mature secretory granules, a second, parallel, pathway was evident involving the nuclear and rough
endoplasmic reticulum (RER)cisternae, but which
bypassed the Golgi complex where activation of
prorenin would be expected to occur (Fig. 3). Thus,
this second pathway would comprise the partially
processed prorenin having additional component(s)
attached, i.e. would be the route for secretion of ‘big
inactive renin’. Moreover, use of this route would
explain why the secretion of ‘inactive renin’ is relatively unresponsive to acute stimulation of renin
secretion. Furthermore, it would be natural to
expect that this route would be amplified in the
FIG.3. Proposal for intracellular biosynthetic pathways leading to the secretion from the juxtaglomerular cell of renin and prorenin.
various situations listed above, in which ‘big inactive renin’ predominates. Part of this would be
contributed no doubt by limitations in the capacity
of granular concentrating mechanisms to cope with
the acceleration in rate of biosynthesis. In addition
the dual pathways might have functional implications in view of the alternative controls: (i) baroreceptor + sympathetic (increasing renin release in
response to decreased blood pressure and volume)
and (ii) macula densa (where stimulation of activatiodsecretion with increased tubular NaCl has
been invoked in the generation of a local reninangiotensin system involved in regulation of
glomerulotubular balance [89,91] and which could
involve mechanisms postulated earlier).
Prorenin has a protease-sensitive activation site
Since discovery of inactive renin which could be
activated by trypsin and pepsin [71], activation has
been demonstrated by proteases of every class
(serine-, aspartyl-, thiol- and metallo-). These
enzymes include glandular and plasma kallikreins,
the arginyl-esteropeptidase (gamma) subunit of
nerve growth factor, acrosin, tonin, plasmin,
cathepsin D and a metalloprotease in Bitis
arietuns venom [103-105, and references cited
therein]. Others have supported these findings and
have shown, in addition, activation by cathepsins B
and H, thrombin and the arginyl-esteropeptidase
subunit of epidermal growth factor. Interestingly,
since cathepsins B and D coexist with renin in
juxtaglomerular cell granules, either may be
involved in activation inside the juxtaglomerular
cell [ 1061. Furthermore, even acid-activation in
plasma, which involves exposure of the active site
through unfolding of intact prorenin [107], requires
participation of the enzyme plasma kallikrein (confirmed by using a probe specific for the active site of
this enzyme [105]) for the activation to be made
irreversible.
Now that the amino acid sequence of human
prorenin is known (from the gene sequence in Fig.
l), activation mechanisms can be examined at the
molecular level. The activation site has the
sequence Mer3-Ly&Arg-’. The fact that this is
identical with the sequence in kininogen [lo81 that
is cleaved by glandular kallikrein and plasma kallikrein (to liberate lysyl-bradykinin and bradykinin,
respectively), together with the fact that this site
would be exposed on the surface of human prorenin
[75] and would therefore be vulnerable to proteolytic attack, could fully account for the ability of
these specialized enzymes to activate prorenin (Fig.
4).This site would also be sensitive to trypsin and
other serine proteases that hydrolyse after dibasic
(Lys-Arg) pairs. As well, cleavage sites for pepsin
New ideasfrom the renin gene
-43
340
1
PRO1
PEPSIN
U
RENlN
TRYPSIN
/ U
7 1 -
Met
U
- Lys -Arg -
u a
1
7
Renal Plasma
KALLlKRElN
FIG.4.Activation site of human prorenin, showing
likely positions for hydrolysis by several enzymes
known to activate 'inactive renin'. Within the juxtaglomerular cell the 'pro' peptide might be hydrolysed at a point located up to 11 residues from the
activation site to give a partially active truncated
prorenin ('inactive renin'). If so, this site could be in
the vicinity of a putative pepsin-sensitive site at - 7.
Since cathepsin D is packaged with renin and has
pepsin-like specificity it could act at this site. The
remainder of the activation peptide may subsequently be hydrolysed by a kallikrein or some other
protease.
can be found in the vicinity and, by comparison
with activation sites for enzymes of other classes,
may serve as a general recognition sequence for the
wide variety of proteases shown to activate inactive
renin.
Conclusion
Therefore the elucidation of the structure of the
human renin gene has provided a sound basis for
speculative thought, from which new concepts will
emerge from on-going experimentation. As well as
reviewing new information concerning the renin
system in extrarenal sites and within cells, this
review predicts that the functional significance of
the extra promoter-like sequences found in the gene
could be linked to the generation of cell-bound
renin by giving rise to alternative precursors with
hydrophilic leader sequences. Next, from gene
sequence and other data, the exact size of the glycosylated prorenin polypeptide is calculated to be MI
44000-46 000. Examination of this in the light of
other information, in order to reconcile a variety of
existing, contradictory data concerning prorenin
and the big form of renin that can be activated by
proteases with little change in M, leads to the conclusion that at least half of the glycosylated prorenin
produced by the juxtaglomerular cell might have
only a portion of the profragment remaining, and an
extra, tightly attached component that is probably
353
polypeptide having a MI of the order of 10000. The
route of secretion of 'big inactive renin' may be via
secretory vesicles which bypass the Golgi region.
Examination of the activation site in the predicted
prorenin sequence explains why so many different
proteases can activate inactive renin.
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