Download Access Slides - Science Signaling

Proteases and Signaling
Sherwin Wilk, Ph.D.
Mount Sinai School of Medicine
Department of Pharmacology & Biological Chemistry
Cell Signaling Systems Course
Phosphorylation and proteolysis are
required in the activation of NF-kB
Traenckner et al, EMBO J. 1994 Nov 15;13(22):5433-5441.
Proteolysis is a hydrolytic reaction
O
O
R2
C
R1
N
+ H 20
R1
C
O
+
R2
NH3+
Classes of proteolytic enzymes:
1.
2.
3.
4.
5.
Serine
Cysteine
Metallo
Aspartyl
Threonine
Protease-Activated Receptors
Fig. 1. Mechanism of G proteincoupled receptor (GPCR) activation by
a reversible binding of a soluble
ligand, such as a neuropeptide, and
irreversible cleavage by a protease.
Déry et al, Am J Physiol. 1998 Jun;274(6 Pt 1):C1429-1452.
Fig. 2. A: protein structure of protein-activated receptor (PAR)-1, PAR-2, and PAR-3. Amino acid sequences in
NH2-terminus and second extracellular loop that are important for receptor activation are shown. Boxed residues
indicate tethered ligand domains (PAR-1, PAR-2, and PAR-3) and anion binding sites (PAR-1 and PAR-3). Arrows
indicate cleavage sites. Bold residues in second extracellular loop are conserved. # Intron/exon border. *
Glycosylation site. B: genomic organization and chromosomal localization of PAR-1 and PAR-2. Both genes consist
of 2 exons and 1 large intron. Exon 1 encodes NH2-terminal domains proximal to cleavage sites, and exon
2 encodes the rest of the receptors. Both receptors are localized within 100 kb on chromosome 5q13.
Déry et al, Am J Physiol. 1998 Jun;274(6 Pt 1):C1429-1452.
TABLE 1
Structure/activity relationships for TRAPs
Peptide
Cell/Tissue Type
Effect (EC50/IC50)
References
SFLLRNPNDKYEPF (TRAP14)
Xenopus oocytes, platelets, rat
aortic rings (endothelium
denuded or intact), guinea pig
gastric longitudinal smooth
muscle, CCL39 hamster
fibroblasts, rat glomerular
mesangial cells, rat astrocytes
4-30 µM
Chao et al., 1992; Coller et al.,
1992; Kawabata et al., 1999c;
Vouret-Craviari et al., 1992; Vu
et al., 1991a; Yang et al., 1992
SFLLR-NH2
Platelets, transfected
mammalian cells, endothelium
denuded and intact RA, CCL39
fibroblasts
0.5-6 µM
Ceruso et al., 1999; Hollenberg
et al., 1996; Kawabata et al.,
1999c; Laniyonu and
Hollenberg, 1995; Natarajan et
al., 1995; Scarborough et al.,
1992
NPNDKYEPF short peptides
(<5)
Platelets
CCL39 fibroblasts, various
species platelets, rat glomerular
mesangial cells, endothelium
denuded RA and gastric LM
>200 µM
Loss of function
Vassallo et al., 1992
Albrightson et al., 1994;
Bernatowicz et al., 1996; Chao
et al., 1992; Connolly et al.,
1994; Vouret-Craviari et al.,
1992
Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.
Substituted peptides
Acetyl-SFLLR-NH2
Platelets, transfected mammalian cells,
rat gomerular mesengial, SH-EP cells
>1000 µM
Albrightson et al., 1994; Coller et al.,
1992; Sakaguchi et al., 1994;
Scarborough et al., 1992
H-SFLLR-NH2
Platelets, transfected mammalian cells,
SH-EP cells
Low activity
Scarborough et al., 1992;
Shimohigashi et al., 1994; Van
Obberghen-Schilling et al., 1993
XFFLR-NH2
Various cell types
Charged amino acids not tolerated,
size/shape important; Thr substitution
yields a PAR-1-specific peptide
Bischoff et al., 1994; Ceruso et al.,
1999; Chao et al., 1992; Hollenberg et
al., 1997; Natarajan et al., 1995;
Sakaguchi et al., 1994; Scarborough et
al., 1992; Van Obberghen-Schilling et
al., 1993; Vassallo et al., 1992; Yang
et al., 1992
SXLLR-NH2
Various cell types
Only aromatic residues tolerated
Albrightson et al., 1994; Ceruso et al.,
1999; Chao et al., 1992; Natarajan et
al., 1995; Nose et al., 1993;
Scarborough et al., 1992; Van
Obberghen-Schilling and Pouyssegur,
1993; Vassallo et al., 1992
SFXLR-NH2
Various cell types
No loss of activity, more active with 3(2-naphthyl)-L-alanine
Bischoff et al., 1994; Blackhart et al.,
1996; Ceruso et al., 1999; Chao et al.,
1992; Laniyonu and Hollenberg, 1995;
Natarajan et al., 1995; Scarborough et
al., 1992; Shimohigashi et al., 1994;
Van Obberghen-Schilling and
Pouyssegur, 1993; Vassallo et al., 1992
SFLXR-NH2
Various cell types
Acidic and basic amino acids not
tolerated
Blackhart et al., 1996; Ceruso et al.,
1999; Chao et al., 1992; Natarajan et
al., 1995; Scarborough et al., 1992;
Vassallo et al., 1992
SFLLX-NH2
Various cell types
Wide range of residues tolerated, but
reduced activity
Blackhart et al., 1996; Ceruso et al.,
1999; Chao et al., 1992; Hollenberg et
al., 1997; Natarajan et al., 1995; Nose
et al., 1998b; Scarborough et al., 1992;
Vassallo et al., 1992
Monocyclic SFLLRN analogues
Platelets
Significant loss of activity
McComsey et al., 1999
Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.
(Table 1 continued)
Improved agonists
S(p-F)FFLRNP
Platelets, SH-EP cells
1.7 µM
Nose et al., 1998a; Nose et al., 1993;
Shimohigashi et al., 1994
S(p-F)FpGuFLR-NH2
Platelets, smooth muscle, mouse
fibroblasts
0.4 µM
Bernatowicz et al., 1996
A(p-F)FRCha-HarY-NH2
Platelets
0.01-0.12 µM
Ahn et al., 1997; Debeir et al., 1997;
Feng et al., 1995; Kawabata et al.,
1999c
A(p-F)FRChaCitY-NH2
Platelets
0.2 µM
Kawabata et al., 1999c
S(p-F)FHarLRK-NH2
Xenopus oocytes
0.05-0.1 µM
Blackhart et al., 1996
S(p-F)F2NaALR-NH2
Platelets, CHRF-288 membranes
80 nM
Seiler et al., 1996
Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.
(Table 1 continued)
Antagonists
IC50
1-phenylacetyl-4-(6-guanidohexanoyl)piperazine
Platelets
50% inhibition of SFLLRNP
Alexopoulos et al., 1998
1-(6-guanidohexanoyl)- 4(phenylacetylamido-methyl)-piperazidine
Platelets
40% inhibition of SFLLRN
Alexopoulos et al., 1998
BMS-197525 {N-trans(p-F)FpGuFLRNH2}
Platelets, smooth muscle,
mouse fibroblasts
0.2 µM of SFLLRNP
Bernatowicz et al., 1996
BMS-200261 {N-trans(p-F)FpGuFLRRNH2}
Platelets
20 nM (SFLLRN) 1.6 µM (thrombin)
Bernatowicz et al., 1996;
Kawabata et al., 1999c
3-Mercapto-propionyl-FChaChaRKNDKNH2
Platelets
0.7-6.4 µM (thrombin)
Kawabata et al., 1999c;
Seiler et al., 1995
LVR(D-)CGKHSR
Rat astrocytes
180 µM ([3H]thymidine incorporation,
by TRAP14 or thrombin)
Debeir et al., 1997
Oxazole-30
Platelets, CHRF
membranes
25 µM (thrombin), 6.6 µM (SFLLRN)
Hoekstra et al., 1998
S(Npys)- Mp-(p-F)F-NHCH(C6H5)2
Platelets
52 µM (SFLLRNP)
Fujita et al., 1999
S(Npys)- Mp-(p-F)F-NHCH2CH(C6H5)2
Platelets
54 µM (SFLLRNP)
Fujita et al., 1999
RWJ-56110 series
Platelets
0.34 µM (thrombin) 0.16 µM
(SFLLRN)
Andrade-Gordon et al., 1999
SCH 79797
Platelets, smooth muscle
70 nM ([3H]haTRAP binding)
Ahn et al., 2000
SCH 203099
Platelets, smooth muscle
45 nM ([3H]haTRAP binding)
Ahn et al., 2000
FR171113
Platelets
0.29 µM (thrombin)
Kato et al., 1999
Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.
(Table 1 continued)
Labels
IC50/Kd
SFp-azido-FLRNPKGGK-biotin
HEL cells
No effect
Bischoff et al., 1994
[3H]haTRAP {[3H]A(p-F)FRChaHarYNH2}
Platelet membranes, platelets
0.15 µM, Kd = 15 nM
Ahn et al., 1997
A(p-F)FRChaHar(125I)Y-NH2
Platelets
0.03 µM
Feng et al., 1995
BMS-200661 {N-trans(pF)FpGuFLROrn}
Platelets
Kd = 10-30 nM
Bernatowicz et al., 1996
SFLLRNPNDKYEPF-biotin
BHK cells
Kd = 3 µmol/l
Takada et al., 1995
BMS-197525, [3H] and biotinylated
derivatives
CHRF-288 cells
Kd = 80 nM or less
Elliott et al., 1999
Amino acid residues X indicates amino acid scan; Har, homoarginine; (pF)F, parafluorophenylalanine; Cha, cyclohexylalanine; N-trans, trans-cinnomoyl;
pGuF, p-guanidino-phenylalanine; Cit, citrulline; Orn, ornithine; 2NaA, 2-naphthylalanine; Npys, S-3-nitro-2-pyridinesulphenyl; β-Mp, β -mercaptopropionyl;
[3H]haTRAP, [3H]A(p-F)FRChaHarY-NH2. All EC50 values refer to platelet aggregation unless indicated otherwise. Likewise, all IC50 values refer to inhibition
of TRAP-induced platelet aggregation unless indicated otherwise.
Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.
(Table 1 continued)
FIG. 2. Structural and functional
domains of PARs. The figure shows
alignment of domains of human
PAR1, PAR2, PAR3, and PAR4. A and
B: mechanism of cleavage and
interaction of the tethered ligand with
extracellular binding domains. C:
functionally important domains in the
amino terminus, second extracellular
loop, and carboxy terminus.
Conserved residues in loop II are in
bold. [Adapted from Derian et al. (89)
and Macfarlane et al. (182).]
Ossovskaya and Bunnett, Physiol Rev. 2004 Apr;84(2):579-621.
FIG. 5. Summary of PAR1 signal
transduction. PAR1 couples to Gi,
G12/13, and Gq11. Gi inhibits
adenylyl cyclase (AC) to reduce
cAMP. G12/13 couples to guanine
nucleotide exchange factors
(GEF), resulting in activation of
Rho, Rho-kinase (ROK), and
serum response elements (SRE).
Gq11 activates phospholipase
C(PLC) to generate inositol
trisphosphate, which mobilizes
Ca2+, and diacylglycerol (DAG),
which activates protein kinase C
(PKC). PAR1 can activate the
mitogen-activated protein kinase
cascade by transactivation of the
EGF receptor, through activation
of PKC, phosphatidylinositol 3kinase (PI3K), Pyk2, and other
mechanisms. G subunits couple
PAR1 to other pathways, such as
activation of G proteins receptor
kinases (GRKs), potassium
channels (Ki), and nonreceptor
tyrosine kinases (TK). [Modified
from Coughlin (68).]
Ossovskaya and Bunnett, Physiol Rev. 2004 Apr;84(2):579-621.
“Cells have capitalized on two highly specific
processes --- phosphorylation and
ubiquitination --- to control complex signaltransduction pathways,” says Maniatis.
“They’ve basically exploited every means of
regulating signaling at their disposal.”
The Ubiquitin-Proteasome
System
Ubiquitin
Ciechanover et al, J Biol Chem. 1980 Aug 25;255(16):7525-7528.
Ciechanover et al, J Biol Chem. 1980 Aug 25;255(16):7525-7528.
E1SH + Ub + ATP
E1S-Ub + PPi + AMP
E1S-Ub + E2SH
ES2-Ub + E1SH
E2-Ub + E3-protein
Ub-protein conjugate + E2 + E3
Pickart, Cell. 2004 Jan 23;116(2):181-190.
The Ubiquitin-Proteasome
System
The Proteasome
Wilk and Orlowski, J Neurochem. 1983 Mar;40(3):842-849.
Groll et al, Nature. 1997 Apr 3;386(6624):463-471.
Wilk and Orlowski, Arch Biochem Biophys. 2000 Nov 1;383(1):1-16.
Ferrell et al, Trends Biochem Sci. 2000 Feb;25(2):83-88.
Tanaka et al, Biochem (Tokyo). 1998 Feb;123(2):195-204.
Impairment of signaling
by proteolysis
Anthrax lethal factor
Composition of anthrax toxin
protective antigen
edema factor
lethal factor
Collier and Young, Annu Rev Cell Dev Biol. 2003;19:45-70.
Figure 1 Stereo ribbon representation of
LF, coloured by domain. The MAPKK-2
substrate is shown as a red ball-and-stick
model, and the Zn2+ ion is labelled. The
RMSD (C) between the cubic and
monoclinic crystal forms is 1.18 ?. The
principal differences lie in the position of
the helical bundle of domain IV, which
undergoes a rigid body shift of ~2 ? relative
to the β-sheet, and a smaller shift of the βsheet of domain I. The third helical element
of domain III is invisible in the monoclinic
crystals, but can be seen in cubic crystals,
albeit with high B-factors. It is possible that
this mobile amphipathic helix has a role in
the membrane-inserting properties of LF
observed in vitro28. Figure prepared with
MOLSCRIPT, RENDER and
RASTER3D29–31.
Pannifer et al, Nature. 2001 Nov 8;414(6860):229-233.
Tonello et al, Nature. 2002 Jul 25;418(6896):386.
Montecucco et al, Trends Biochem Sci. 2004 Jun;29(6):282-285.
Inhibition of signaling by the
Yersinia effector YopJ
Fig. 1. Profile of YopJ inhibition. YopJ is delivered into the target host cytosol via a type III secretion system. YopJ
blocks activation of the superfamily of MAPK kinases, including MKKs (which activate the MAPK pathways), and
IKKβ (which activates the NFκB pathway). The inhibition results in the inability of the cell to produce cytokines
and anti-apoptotic machinery. The possibility exists that YopJ may directly activate the cell death machinery, as
denoted by the red arrow with the question mark.
Orth, Curr Opin Microbiol. 2002 Feb;5(1):38-43.
Fig. 2. Amino acid sequence alignment of AVP, the family of YopJ homologues and Ulp1. The figure shows
alignment of the catalytic core of cysteine proteases with the catalytic triad (denoted in red) and identities or
similarities (outlined and shaded light blue). The alignment includes (protein accession numbers in
parentheses): AVP (2781331); YopJ (P31498); AvrA (AAB83970); AvrBst (AAD39255); AvrRxv (AAA27595);
AvrXv4 (AAG39033); AvrPpiG1 (CAC16700); ORF5 (AAF71492); ORFB (AAF62400); Y4LO (P55555); and
Ulp1 (NP_015305).
Orth, Curr Opin Microbiol. 2002 Feb;5(1):38-43.
Fig. 3. Modification of proteins by
ubiquitin or SUMO. Ubiquitin and
SUMO are linked to target proteins
by an isopeptide bond. The carboxyl
terminus of these modifying proteins
(blue) is conjugated to the -amine of
a lysine residue (LYS) in the target
protein. Ubiquitin, unlike SUMO, can
conjugate more monomers to itself to
form a polyubiquitin chain. Deubiquitinating enzymes (DUB) and
ubiquitin-like protein proteases (for
example, Ulp1) can cleave the
isopeptide bond in the ubiquitinconjugates (green arrows) or SUMOconjugates (pink arrow), respectively.
Orth, Curr Opin Microbiol. 2002 Feb;5(1):38-43.
Fig. 4. Similarities between two reversible post-translational modifications. Phosphorylation is shown on
the left and ubiquitination is shown on the right. Addition of phophorylation (P) by kinases and ubiquitin
(Ub) by the E1–E2–E3 conjugation machinery to target proteins is shown in blue. The requirement for
energy is noted by the purple ATP. The removal of phosphorylation by a phosphatase or of ubiquitin by a
de-ubiquitinating enzyme is shown in red. The target protein (green) can cycle from an unmodified to a
modified state. The model can be used for ubiquitin-like proteins by replacing the modification with, for
example, SUMO or NEDD8, and replacing the protease with a ubiquitin-like protein protease.
Orth, Curr Opin Microbiol. 2002 Feb;5(1):38-43.
Regulated Intramembranous
Proteolysis
Notch Pathway
Mumm and Kopan, Dev Biol. 2000 Dec 15;228(2):151-165.
Fig. 2.
Presenilin and presenilin-like MpMPs regulate
cleavage of diverse substrates. IP by secretase (presenilin) requires prior JP by
either ß-secretase (shown) or -secretase
(not shown). The combination of ß- and secretase cleavages produce the Alzheimer's
disease-associated peptide Aß, whereas the
combination of - and  -secretase cleavages
produce a peptide known as P3 (not shown),
whose role in the disease process in currently
unknown. Both sequential cleavage events
produce CTF [also known as APP
intracellular domain (AID or AICD)], which can
translocate to the nucleus to form a
transcriptionally active complex with Fe65 and
Tip60. The nuclear targets of the CTF are not
known. Although a single -secretase
cleavage is shown, -secretase cleaves the
transmembrane domain of APP at multiple
sites both near the middle of the membrane
and near the cytoplasmic face of the
membrane.
Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.
Fig. 3.
The presenilin -secretase appears to
be an aspartyl MpMP. The eighttransmembrane domain model of PS is
shown. Although the topology of the first
six transmembrane domains is
accepted, the exact topology of the
COOH-terminal transmembrane
remains controversial. The red and
orange stars indicate the location of the
conserved active site residues within the
transmembrane domains 6 and 7.
Mutation of either of these aspartates
(D) results in a dominant-negative PS
that inhibits -secretase activity. A red
triangle also indicates the location of the
evolutionarily conserved Pro-Ala-Leu
(PAL) motif essential for PS stability.
The box to the right shows the
consensus sequences surrounding
these conserved residues. The putative
location of these active site aspartates
places them in location to cleave near
the COOH-terminus of Aß. Y, Trp; G,
Gly.
Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.
Fig. 4.
-Secretase also cleaves the type
1 surface receptor Notch and its
family members. Following binding
of a ligand present on the surface
of the adjacent cell (delta, serrate,
or Lag-2) to the extracellular
domain of Notch, a conformational
change occurs permitting JP by the
metalloprotease disintegrin
(ADAM17). The Notch COOHterminal fragment (NEXT) is then
cleaved by  -secretase to release
the Notch intracellular domain
(NICD) into the cytoplasm. Upon
release, the NICD translocates to
the nucleus, where it acts as a
transcriptional regulator through its
interactions with the transcription
factor CSL.
Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.
Regulated Intramembranous
Proteolysis
Sterol regulatory element binding
protein (SREBP)
Fig. 1. (A) RIP of SREBP involves an
additional protein, SCAP, that regulates
SREBP transport. SREBP exists in a hairpinlike conformation with a small luminal loop
and two large cytoplasmic domains. The NH2terminal domain is a basic helix-loop-helix
(bHLH) transcription factor and the COOHterminal domain is a regulatory factor (REG)
that binds SCAP. When cells are loaded with
sterols, the complex of SCAP and SREBP is
sequestered in the ER. Upon sterol
deprivation, the SCAP-SREBP complex is
transported to a post-ER compartment
(thought to be the cis or medial Golgi) where
S1P-mediated juxtamemembranous
proteolysis (JP) of the small luminal loop of
SREBP occurs. The bHLH domain of SREBP
is then translocated to the nucleus, where it
binds to sterol regulatory elements and
controls transcription of a number of genes
involved in sterol metabolism. (B) S2P
appears to be a metalloprotease-type
intramembranous cleaving protease (MpMP).
A schematic of the overall topology of S2P is
indicated. The position of residues involved in
catalysis is indicted by the stars. The
conserved residues indicated by these stars
are shown in the box. The conserved active
site histidines (red star) appear to be located
at a site in the membrane that permits
cleavage of SREBP within its transmembrane
domain close to the cytoplasmic face of the
membrane. These residues, along with the
remote aspartate, are hypothesized to form
an active site similar to that formed by classic
metalloprotease. The true three-dimensional
structure of S2P is not known, and location of
residues is strictly based on hydropathy plots
and topology studies.
Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.
Regulated Intramembranous
Proteolysis
Rhomboid and EGF signaling
Figure 2. Identification of Putative Catalytic Residues
of Rhomboid-1(A) Conserved residues were
individually mutated to alanine and the ability of the
mutant proteins to mediate Spitz cleavage was
determined. The upper panel shows Western blots of
cleaved GFP-Spitz in the medium of cells transfected
with GFP-Spitz, Star, and mutant or wild-type
Rhomboid-1. Mutation of W151, R152, N169, G215,
S217, and H281 abolished detectable Rhomboid-1
activity—compare with no Rhomboid-1 control (−). The
lower panel shows Rhomboid-1 levels in cells,
assessed using N-terminally HA-tagged Rhomboid-1
mutants.(B) All mutants that were unable to mediate
Spitz cleavage were, like the wild-type protein,
localized to the Golgi apparatus. HA-tagged S217A in
a COS cell is shown in green; anti-p115 (Transduction
Labs), a Golgi marker, in red.(C) Comparison of the
conserved GASGG motif surrounding the putative
Rhomboid-1 active serine (S217) with that of serine
proteases (family S1A according to MEROPS
classification). The subscripts represent the
percentage conservation at each residue (total
nonredundant sequences for each family are reported
as "n" values).(D) Rhomboid-1 with conservative
mutations S217C and S217T did not catalyze Spitz
cleavage above background levels; panels as in
(A).(E) Diagram of Rhomboid-1 residues essential for
Spitz cleavage; conserved but nonessential residues
are shown in green, essential residues in red. The
TMD predictions were agreed by a variety of
algorithms, including TMHMM and TMPred (available
at http://ca.expasy.org/tools/#transmem)
Urban et al, Cell. 2001 Oct 19;107(2):173-182.
Figure 7. Model of
Rhomboid-1 ActionWe
propose that the conserved
asparagine (N), histidine (H),
and serine (S) form a serine
protease catalytic triad that
hydrolyses the Spitz
polypeptide within its TMD
(although the role of the
asparagine is uncertain—
see text). Our data imply
that this occurs
approximately one-third of
the distance into the
membrane bilayer from the
lumenal surface. The
lumenal tryptophan (W) and
arginine (R) are also
essential (although the
tryptophan appears less
critical in human RHBDL2),
but their function in the
catalytic mechanism
remains to be determined
Urban et al, Cell. 2001 Oct 19;107(2):173-182.
Fig. 7.
RIP by a novel serine MpMP, Rhomboid. (A)
Drosophila Rhomboid-1 promotes cleavage
of at least three epidermal growth factor
(EGF)-like type 1membrane proteins, Spitz,
Keren, and Gurken. Rhomboid cleavage
does not appear to require prior JP. It also is
unique in that it releases the ectodomain
from the membrane rather than releasing a
cytoplasmic domain. In their uncleaved
forms, these EGF-like ligands are
sequestered in the ER. In activated cells, the
transmembrane protein Star facilitates
trafficking to the Golgi where the active
Rhomboid protease resides. In the Golgi the
ligands are cleaved near the luminal border
of their transmembrane domains, releasing
them from the membrane. Once released,
the ligand is secreted where it binds the
EGFR. (B) The predicted topology of
Drosophila Rhomboid-1. Rhomboids are
serine MpMPs. The stars show the location
of the putative catalytic triad and the box
describes these consensus sequences (N,
Asn; G, Gly; A, Ala; S, Ser; H, His). As is the
case for other MpMPs, the active site
residues appear to be positioned in an
appropriate location to carry out their
cleavage, which in this case occurs near the
luminal face of the membrane. In addition,
the red triangle shows the location of
another sequence (W, Trp; R, Arg) essential
for function.
Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.