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CHC22 and parietal cells

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CHC22 and parietal cells

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University of Southern California
CHC22 and Parietal
Cells
Thesis submitted to the graduate school for the partial fulfillment towards
M.S. in Pharmaceutical Sciences. The degree conferral date – August 2017
Anuj Jamenis

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Table of Contents
Chapter 1 Abstract 3
Chapter 2 Introduction 4
2.1 Clathrin 4
2.2 CHCs 5
2.2.1 CHC17 6
2.2.2 CHC22 9
2.3 CLCs 11
2.4 Mechanism of Clathrin Lattice Formation 13
2.5 Clathrin Functions 18
2.5.1 TGN and Golgi Functions 19
2.5.2 Endosomal Sorting 20
2.5.3 Endocytosis, Phosphorylation, and Signaling 21
2.5.4 Storage compartment formation 21
2.5.5 Other functions 22
2.6 Gastric Acid Secretion and Parietal Cells 24
2.7 H,K-ATPase 25
2.8 Acid secretion associated membrane dynamics 27
2.9 Inhibition of Acid secretion 28
Chapter 3 Materials and Methods 30
3.1 Sample Preparation 30
3.2 Antibodies 31
3.3 Human Skeletal Muscle Lysate 31
3.4 Western Blot 31
3.5 Coomassie Blue 32
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Chapter 4 Results 33
Chapter 5 Conclusion and Future Direction 43
References 45

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Chapter 1: Abstract
Clathrin is considered to be the prototype vesicle coat protein whose self-assembly
mediates sorting of membrane cargo and recruitment of lipid modifiers to help deform the
membrane into a budding vesicle. Detailed knowledge of the biochemistry, structure, and
interacting proteins of the ubiquitous CHC17 isoform of clathrin has accumulated since its first
observation, isolation, and characterization, almost 50 years ago, but the characterization of the
role of the recently-discovered CHC22 isoform of clathrin is still in a nascent stage. Although
CHC17 has well defined roles in multiple cellular processes, the most prominent documented
function of CHC22 remains its modulation of GLUT4 glucose transporter. Similarly the modulation
of trafficking of the gastric H,K-ATPase by vesicle coat proteins is not clearly understood. Here
we investigate the hypothesis that CHC22 may be involved in the regulatory process of H,K-
ATPase, due to the similarities in the GLUT4 and H,K-ATPase trafficking. Western blotting has
shown that CHC22 is in fact present in the gastric parietal cells of hog and rabbit. There remain a
number of unanswered questions that require further investigation.

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Chapter 2: Introduction
2.1 Clathrin
Clathrin is involved in endocytosis and exocytosis, facilitating the formation of vesicles
allowing cells to communicate, transfer nutrients, internalize receptors, mediate immune
responses and clean up the debris in the cellular and tissue environment. Clathrin was first
isolated and named by Barbara Pearse in 1975 as a newly identified coat-protein
1,26
. A coat
protein is a protein that is used by the cell to form small vesicles by forming a polyhedral lattice
that surrounds the vesicle into which cargo molecules for endocytosis or exocytosis are packaged.
Clathrin is an approximately 190 kDa coat-protein, also known as the heavy chain, that
forms homologous trimeric structures called triskelia which in turn form a lattice. Each 190 kDa
heavy chain combines with an approximately 25 kDa light chain and then with two other heavy
chains (along with their associated light chains) to form a triskelion. These triskelia then interact
with each other forming a polyhedral lattice that surrounds vesicles. The three heavy chains
provide the structural backbone of the clathrin lattice, and the associated three light chains are
thought to regulate the formation and disassembly of a clathrin lattice by allowing for the
interaction of other proteins with the lattice (see Figure 1).
1
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Figure 1 (A) Assembled clathrin at the plasma membrane showing the triskelia (red) in relation to the flat
lattice of the clathrin coat on the inner (cytoplasmic) side of the cell membrane and a forming sphere where a CCV
is about to separate from the membrane surface.
1
(B) A hypothesized rendering of a CCV showing the polyhedral
lattice formed by clathrin heavy chains and some cargo incorporated inside. Clathrin light chains and the membrane
of the vesicle are not pictured.
11
Clathrin subunits are encoded by various genes on the human chromosomes, as shown in
Table 1.
Table 1: Chromosomal locations of clathrin components
1

2.2 CHCs
Clathrin heavy chains (CHC) form the backbone of the triskelion molecule. In humans,
two isoforms of CHCs are present, namely, CHC17 and CHC22.
(A)
(B)
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The isoform found on chromosome 17 is called CHC 17 (Clathrin Heavy Chain 17). CHC17
is ubiquitously expressed in all nucleated mammalian cells throughout the body and is involved
in the regulation of many intracellular membrane traffic pathways including receptor-mediated
endocytosis, lysosome biogenesis, and endosomal sorting.
1,2
Clathrin is involved in accumulating
target proteins which are to be internalized or transported among intracellular organelles. The
accumulation of the target proteins at specific sites on cellular membranes then leads to a
stepwise budding or internalization process involving recognition by clathrin adaptor proteins
and CHC, polymerization, and finally removal of clathrin molecules by specific enzymes once the
vesicle is formed (explained in detail in section below on mechanism of lattice formation).
The other form of CHC is CHC22 found on chromosome 22. This form is present primarily
in skeletal muscle cells and adipose cells and is involved in regulation of GLUT4 glucose
transporters, but CHC22 has not been convincingly shown to be involved in endocytosis.
4

2.2.1 CHC17
CHC17 is the most common isoform of clathrin heavy chain and is found in all nucleated
cells in humans. It is expressed by the gene CLTC on chromosome 17. It is a highly conserved gene
found in most eukaryotes. The structure of the clathrin heavy chain is often described as a leg,
with subdomains representing the foot (the N-terminal domain), followed by the ankle, distal leg,
knee, proximal leg, and trimerization domains
1
(see Figure 2). The CHC17 monomer, which self-
assembles into triskelia, is composed of 2 regions
1
. The first region, alpha-solenoid region, has 8
repeats of 10 helices of 10-12 residues connected by loops, forming a super-helix of short alpha
helices. The proximal leg domain is composed of smaller structural modules referred to as
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clathrin heavy chain repeat motifs (CHCR) which are numbered from 0 to 7. The light chains bind
primarily to the proximal leg portion of the heavy chain up through CHCR7 between the residues
1198 and 1575, more specifically cysteines 1565, 1573, and 1569 near the trimerization domain.
Cysteine 1528, present near the boundary between proximal leg and trimerization domain,
inhibits trimerization when CLCs are not bound, thereby inhibiting unnecessary aggregation of
clathrin heavy chains. However, the isolated heavy chain trimers can reassemble into regular
polygonal cage structures in vitro in the absence of light chains. Thus, while CHCR7 contributes
toward clathrin triskelion trimerization, CHCR0 connects the alpha-solenoid region to the second
region of CHC, called the “beta-propeller domain” (see Figure 2)
1
.
The N-terminal domain of CHC17 consists of a seven-bladed β-propeller structure, that
contains multiple binding sites for interaction with other proteins
1
known to assist in the
regulation of vesicle formation
1,4
The β-propeller’s main interactions are with clathrin adaptor
proteins such AP-1 and AP-2, as well as accessory proteins. One of the β-propeller -binding motifs
on proteins such as amphiphysin, called the clathrin box, is a clathrin-box motif (CBMs) of the
sequence LΦXΦ[DE] (single amino acid code, where Φ is a bulky hydrophobic residue and X is
any amino acid, but is often polar). The interaction of the CBM with the β-propeller is also the
target for the clathrin chemical inhibitors Pitstop 1 and Pitstop 2
1,16
.
The structural view at 10-12 Å resolution has indicated that clathrin basket lattice
assembly is dependent on the interaction between helical faces and loop faces of CHCRs from
two proximal and two distal leg regions on two triskelia that meet at one edge
1
. There are also
additional binding sites for clathrin adaptors between CHCR1 and CHCR2 which are indicated to
be involved in lattice formation.
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Figure 2 Structural features of clathrin heavy chains. The domain structures and amino acid boundaries
are indicated for functional domains of CHC17. The structures reproduced are from PyMol (triskelion-accession
number 3IYV; trimerization domain-accession number 3LVH; terminal domain-accession number 2XZG; proximal leg-
accession number 1B89) and are based on Fotin et al. (2004b), ter Haar et al. (1998), and Ybe et al. (1999). The four
numbered sites on the terminal domain structure represent binding sites for interacting proteins based on Lemmon
& Traub (2012). At the bottom, the predicted domain structure of CHC22 is aligned with CHC17, and the amino acid
boundaries of the eight clathrin heavy chain repeats (CHCR 0–7) in CHC17 are delineated. The red marks highlight
differences between the CHC17 and CHC22 protein families determined by DIVERGE analysis, adapted from
Wakeham et al. (2005).
1

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2.2.2 CHC22
CHC22 was first discovered during the gene identification and mapping studies of human
chromosome 22. The gene encoding CHC22 was then called CLTCL-1 and was found to encode a
protein very similar to the known clathrin heavy chain protein (CHC17) encoded by the gene CLTC
on chromosome 17.
5
The main difference was found to be in the carboxyl terminal of the new
protein (see Figure 2). It was also found to be ubiquitously expressed in fetal tissues but only
selectively in adults, namely skeletal muscle cells.
8
It was further discovered that the CHC22 was
involved in the internalization of GLUT-4 glucose transporters
8
.
The GLUT-4 glucose transporters are involved in the absorption of glucose. They are found
primarily in adipose tissue and striated muscle cells. They are regulated by insulin. In conditions
of low insulin, the GLUT-4 glucose transporters are sequestered in intracellular vesicles (see
Figure 3). This process is carried out by the CHC22 in a manner thought to be similar to the
process of CHC17-mediated internalization of other receptors.
CHC22 has also been shown to be extremely important in neural development during fetal
development. Lack of CHC22 during this stage is associated with a number of developmental
disorders, such as DiGeorge syndrome and velocardiofacial syndrome
1,7
. CHC22 is also shown to
be important in the development of human pain and touch
6
. The novel disorder is a congenital
recessive disorder characterized by cognitive delay, inability to feel touch, congenital insensitivity
to feel pain. This is caused by a missense mutation in the CTCL-1 gene resulting in mutated CHC22
but not a reduction in expression levels of CHC22
6
.

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CHC22 has been shown to have a role in the skeletal muscle regeneration to an extent
that the mouse knockout model of CHC22 has extremely curbed regeneration of damaged muscle
cells.
4

Figure 3 (A) Schematic diagram from a 1977 paper by Anderson, Goldstein and Brown, showing how the
LDL receptor might use an ‘internalization site’ to become incorporated into a coated pit.
20
(B) The accumulation of
receptor (green) at the cell membrane leading to recruitment of clathrin triskelia (yellow) followed by successive
steps leading to formation of a CCV and a cytoplasmic vesicle that will fuse with an early endosome.
Despite all of these functions the interesting discovery was that mice, wild or lab grown,
only possess a pseudogene of CHC22
37
. In fact it was found that introducing an active CHC22
gene in mice resulted in the mice exhibiting diabetes-like symptoms, thought to be because of
disruption of normal glucose clearing pathways. One possibility may be that mice may not need
CHC22 because they do not rely as much as humans do on their muscle cells to clear glucose from
(A) (B)
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the blood. In humans, skeletal muscle cells account for 90% of this job, but in mice, the liver does
the majority of the work.
37,8
2.3 CLCs
The CHC17 monomers are not found in the cell but rather are present assembled as
trimers and each CHC is bound to one clathrin light chain (CLC). The mammalian CLCs are
comprised of two isoforms, CLCa and CLCb, also called LCa and LCb respectively. LCa is primarily
found in brain tissue while LCb is primarily found in the lymphatic system
1
. The binding of LCs to
CHCs has been long thought to be involved in the regulation of basket lattice formation by
clathrin. The CLCs can be bound to CHCs in two conformations, “straightened” and “bent” (see
Figure 4). The bent conformation is compatible with lattice formation while the straight
conformation is not. Thus the change of conformation of LCs has been hypothesized to contribute
towards the regulation of lattice formation by bending the triskelion.
Radiolabeled light chains can be shown to bind with high affinity (Kd < 10
-10
M) to heavy
chain trimers, to heavy chain cages, or to a 110,000 Da tryptic fragment of the heavy chain. In
addition, both light chains compete with each other for the same binding sites on heavy chains.
Yeast, on the other hand, produces only one isoform of CLC called Clc1. This has been
shown to play the role similar to mammalian Clcs but the process may be more efficient since the
competition between LCa and LCb is absent.
15

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Figure 4 Projected structures of triskelia with all bent or all straight knees. Diagrams on the right show how the
straight knee conformation is incompatible with the dimensions of the clathrin lattice, whereas the bent knee
resulting from a conformational change of CLC is compatible with lattice assembly. Panels b and c reproduced from
Wilbur et al. (2010b).
1

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2.4 Mechanism of Clathrin Lattice Formation
Clathrins have varied functions being involved in cellular membrane trafficking pathways,
signaling pathways, regulation of receptors and transporters by forming storage compartments,
and they are also involved in trans-Golgi network (TGN) functions. Clathrins are also involved in
actin organization, viral invasion of cells, and mitosis.
10,12

The central dogma of any task that is performed by clathrins is their ability to identify and
package specific proteins into a forming transport vesicle. Clathrin is hypothesized to get
recruited at the appropriate cellular membrane by adaptor proteins, such as AP-1 and AP-2. AP-
1 and AP-2 attach to the clathrin box motif of triskelia through their unstructured hinge region
and the alpha, beta, and gamma appendage domains
1,11
(see Figure 5).
Clathrin also binds to the monomeric proteins known as Golgi-localized, gamma-adaptin
ear-containing, ARF-binding protein (GGA). Members of this family are ubiquitous coat proteins
that regulate the trafficking of proteins between the TGN and the lysosome.
21
AP-1, AP-2, and
GGAs also have various other motifs that recognize specific regions on target proteins or
receptors (in this case the proteins to be encapsulated by clathrin) like the VPS-27, Hrs and STAM
occurring domain (VHS domain), an approximately 140 residue long domain commonly found in
the N-terminal region of a number of proteins, and which is also present on GGAs; and interact
with the DXXLL motif (D-aspartic acid, X-any amino acid L-dileucine) which is present on the cargo
proteins
38
. Many of these adaptors also bind multiple accessory proteins such as vacuolar protein
sorting-associated protein (VPS) family proteins that include VPS28, VPS37, that help enhance
the lipid and cargo binding abilities of the adaptor proteins. They recruit membrane-deforming
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proteins that contain domains which are named after three proteins they were found in: Bin,
Amphiphysin and Rvs
35
(BAR domain). BAR domains are highly conserved protein dimerization
domains that occur in many proteins involved in membrane dynamics in a cell. The BAR domain
is banana-shaped and binds to membranes via its concave face
35
. Adaptor proteins also recruit
multiple kinases and phosphatases.

Figure 5 (A) A cartoon showing the flexible appendage of AP-1 and AP-2 which have clathrin box motifs that
allow them to bind to clathrin. (B) The hinge region of -adaptin which forms the flexible appendage along with its
binding site to clathrin.
(B)
(A)
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Unlike AP-1 and AP-2, the additional adaptor proteins AP-3, AP-4, and AP-5 are not
essential for viability in animals
1
. There are naturally occurring mutations in AP-3 in Drosophila
and mice
11
, as well as in humans with Hermansky-Pudlak syndrome, a disorder mainly affecting
lysosome-related organelles
11
. There are also patients with mutations in AP-4 or AP-5, both of
which cause hereditary spastic paraplegia
1
. It is not clear precisely why. Recently a family of
proteins was identified called Clathrin-Associated Sorting Proteins (CLASPs), and have been
demonstrated to have all the functions of AP-1, AP-2, and GGAs
1
. Interestingly, however, AP-3,
AP-4, and AP-5 lack one of the four functions (clathrin binding, cargo binding, lipid binding, and
accessory protein binding) ascribed to CLASPs.
11

The basic steps involved in clathrin vesicle formation are (see Figure 6):
a. Recognition of target proteins by adaptor proteins
b. Polymerization of coat lattices
c. Recruitment of dynamin
d. Pinching off (a function of dynamin)
e. Removal of clathrin from the CCV formed so that the endocytic vesicle can fuse
with early endosomal compartments
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Figure 6 Proposed steps in the formation of an endocytic CCV. A) AP-2 complexes are recruited to the
plasma membrane by the α subunit binding to PIP2 (red), but rapidly cycle on and off. B) Electrostatic interactions
between PIP2 and the μ subunit may open up the complex and expose additional binding sites. C) Interactions with
cargo (orange and yellow) further stabilize the open conformation of AP-2 and enable the coated pits to mature
more efficiently D) Clathrin also stabilizes the association of AP-2 with the plasma membrane: one triskelion
recruited to two AP-2 complexes increases the residence time of the AP-2 at the plasma membrane and results in
the recruitment of more clathrin and AP-2. E and F) The coated patch (now shown at lower magnification) keeps
growing, incorporating various early-arriving alternative adaptors, such as CALM and the muniscins FCHo1/FCHo2
(grey-green). G) Dynamin (purple) is recruited to the neck of the deeply invaginated coated pit to facilitate scission
from the plasma membrane. H) Immediately after scission, uncoating machinery is recruited, including auxilin
(orange) and OCRL1 (blue). The entire sequence of events, from (A) to (H), takes about 2 min.
11

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The last step of the process, i.e. removal of clathrin coat molecules is very important as in
presence of clathrin the CCVs cannot fuse with other membranes, thus, for example, if clathrin is
not removed from the outer surface of the vesicle, cargo from the endocytic vesicle cannot be
delivered to the early endosome. There are various regulatory proteins involved in this process
such as beta-arrestins which are involved in the physical separation of receptor and its ligand
prior to sequestration by clathrins, or the multiple kinases involved in activation of APs. It has
been demonstrated that during endocytosis the alpha and the beta subunits of both AP-1 and
AP-2 are dephosphorylated in the cytosol by as a yet unknown phosphatase (except for
phosphatase 2A which is associated with CCVs and can dephosphorylate the serine residues) to
facilitate the clathrin binding. At the cargo binding sites, kinases like AAK1 which phosphorylates
threonine 156 of the μ2 subunit of AP-2, enhancing its cargo binding ability, and this activity has
been shown to be stimulated by clathrin
27
. Similarly, the clathrin binding kinase CVAK104 is
known to be present in the endocytic pathway and TGN, and it influences protein traffic of the
membrane-fusing protein SNARE protein complex
28
. Dephosphorylation of the μ2 subunit of AP2,
which reduces its cargo affinity, contributes to CCV uncoating and is stimulated by displacement
of AAK1 from AP-2 through competition by the Rab5 GEF RME-6.
1

Clathrins may also be directly modulated by additional phosphorylation pathways such as
the CHC17 phosphorylation at tyrosine 1477 and 1487 by non-receptor tyrosine kinases from Src
family kinases in ligand-gated, receptor-mediated endocytosis
33
. This regulation is seen in T-cell
receptor and B-cell receptor uptake
33,1
.
Another protein involved in the regulation of endocytosis is dynamin which is involved in
the third step of CCV formation. It helps to seal the immature vesicle being formed at the cell
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surface by closing and ligating the two ends of the vesicle to each other and ligating the cell
membrane back together to ensure no damage to the cell membrane. Dynamins are self-
assembling large GTPases of ∼100 kDa that interact with membranes and form ringed structures.
They are members of a larger family of dynamin-like proteins that participate in membrane
fission at multiple intracellular sites. Dynamin assembles at the neck of a budded endocytic CCV
in neuronal and non-neuronal cells. Subsequent hydrolysis of GTP induces a conformational
change that promotes vesicle scission or pinching off. Although dynamins are primarily implicated
in endocytotic pathways, it is thought today that they may also participate in TGN pathways but
may not be essential for them.
11

2.5 Clathrin Functions
As seen in the section above clathrins major function is the formation of CCVs as part of
either endocytotic pathway or for transport from and return to the TGN. CCVs have been
implicated in the uptake of numerous transporters and receptors including GLUT-4, Tf, and
GPCRs. The uptake process has been explained above; however, clathrin has been implicated in
the regulation of other cellular trafficking pathways and other cellular functions, which are
described in the following section.
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Figure 7 Clathrin and its various functions. The functions and pathways are described in the text that follows.
1

2.5.1 TGN and Golgi Functions
Clathrin has multiple well-defined roles at the TGN (see Figure 7). The TGN is the final
cisternal structure in the Golgi complex, from which proteins are packaged into vesicles destined
to lysosomes, secretory vesicles, or the cell surface. The TGN is usually positioned adjacent to the
stacks of the Golgi apparatus, but can also be separate from the stacks. The TGN may act as an
early endosome in yeast and plants.
17
Clathrin also plays a role in TGN-to-lysosome sorting
36
.
Clathrin heavy chain 17 has recently been shown to play a role in Golgi-complex integrity.
Its depletion by siRNA impairs the reassembling of Golgi after drug-induced and mitotic
fragmentation.
18,19
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2.5.2 Endosomal Sorting

Clathrin has been hypothesized to participate in pathways of endosomal sorting and rapid
recycling of cargo from early endosomes
2
. This function has been widely debated since we lack
the precision tools required for interfering with the clathrin function, and it is thought that the
clathrin-interfering drugs like “pitstops” may be disrupting the delicately balanced pathways of
the cell
1
. It has been shown that for cargo to be enclosed in tubules it is associated with clathrin-
coated patches which are colocalized with tubule forming components like TGN38 and TGN46
39
.
However, there is evidence of a so-called G-clathrin colocalizing with GGA1 distinct from late
endosomal and lysosomal pathways and may be involved in receptor recycling
32
. G-clathrin are
structures characterized by localized but extremely rapid movement, leading to the hypothesis
that they are coated buds on waving membrane tubules. G-clathrin structures have structurally
and functionally distinct features. They lack detectable adaptor proteins AP-1 and AP-2 but
contain GGA1 [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding
protein] as well as the cation-dependent mannose-6-phosphate receptor.
32
While they
accumulate internalized transferrin (Tf), they do not contain detectable levels of cargos targeted
for the late endosome/lysosome pathway such as EGF and dextran.
32

Clathrin is also involved in sorting cargo for TGN via retrograde pathways 2 and 3 (see
Figure 7). Clathrin is involved in possibly both pathways through AP-1 and GGA2 binding. Clathrin
contributes to the mannose-6-phosphatase receptor and Shiga toxin sorting.
2
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Clathrins have also been shown to contribute to sorting in the multivesicular bodies
formed by ESCRT proteins
2
. CHC17 is recruited by ESCRT0 component in a bilayered coat. This
interaction has been proposed to cause cargo capture.
2
2.5.3 Endocytosis, Phosphorylation, and Signaling
Clathrin appears to be increased at the plasma membrane in phosphorylation promoting
cytoplasmic conditions. This lead to the hypothesis that CHC17 phosphorylation temporarily
inhibits budding of CCVs and stabilizes clathrin-coated pits. This was further confirmed by the
observation that at the location where SRC-family kinases are activated, there are more
observable clathrin-coated pits.
1,11

Receptor internalization can be regulated by multiple pathways, altering cytoplasmic tail
recognition by ligand binding as seen in epidermal growth factor (EGF) receptors
31
or AP-2
recruitment by beta-arrestin because of its phosphorylation due to ligand binding as seen in
GPCRs.
1,11

The clathrin-mediated uptake of receptors is also implicated in fine-tuning the regulation
of receptor signaling. For careful synchronization of endocytosis, CCV formation could enhance
or attenuate signaling by either enriching the receptors on the membrane or remove them
completely, respectively.
1,11

2.5.4 Storage compartment formation
CHC22 has increased expression in myoblasts and adipocytes. It plays a tissue-specific role
in these cells for the late endosome to TGN retrograde sorting pathway. This pathway is
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important for the sorting of GLUT4 glucose transporters to GLUT4 storage compartments (GSCs),
which are insulin responsive. CHC22 knockout rats are unable to form GSCs.
4,8
Upon stimulation by insulin, GSC fuse with the cellular plasma membrane of muscle cells
and adipocytes, therefore delivering GLUT4 to the cell surface, where it can clear glucose rapidly
from blood plasma and import it into muscles and fat.
8
While CHC22 plays no apparent direct
role in activation of GLUT4, on the removal of stimulus, CHC22 sorts GLUT4 into the GSCs. The
CHC22 sorting step has been shown to be very important in type 2 diabetes patients.
8
These
patients are unable to exocytose the GLUT4 from their GSCs, and thus they are termed as insulin-
resistant. It is hypothesized that this is caused by excessive CHC22 recruitment at GSCs making
them unable to release GLUT4 upon stimulation by insulin
4,8
. An accumulation of APs and GGA
adaptors on GSGs was also observed. Studies with mice in which CHC22 was transgenically
introduced into their muscle cells and adipocytes have been shown to exhibit symptoms similar
to those of type 2 diabetic patients, including the increase of GSCs and elevated blood glucose
levels, that are observed in patients.
4,8
2.5.5 Other functions
Huntington-interacting proteins (Hip) that bind to CLCs mediate the interaction between
clathrin and actin
30
. Through this interaction, clathrin plays a role in actin organization. The
clathrin lattice is thought to influence the organization of actin at larger membrane interfaces
during viral and bacterial internalization and infections
1
. In yeast, actin counteracts the
membrane tension generated due to clathrin-mediated endocytosis
30
. It plays a similar role when
artificial tension is applied to mammalian cells
30
. This activity is again modulated by
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phosphorylation of Hip
30
. Even in normal cells (without artificial membrane tension) actin has
been consistently shown to be present at the ‘neck’ of forming CCVs. The actin organizing
complex protein Cytoplasmic FMR1-interacting protein (CYFIP) has been shown to interact with
CHC17 at the ends of coated tubules in TGN, and this may contribute to the actin-based extension
of tubules.
1,11,31
CHC17 has also been to form a TACC3-ch-TOG-CHC17 complex which plays a role in cell
cycle.
3
Its attachment to the TACC3 protein depends on TACC3 phosphorylation by Aurora-A
kinase which is activated at the onset of mitosis.
10,12
The TACC3-ch-TOG-CHC17 complex stabilizes
kinetochore fibers which function in segregation of chromosomes. Kinetochores are composed
of bundled microtubules. In the absence of CHC17, the protein bridges in these microtubules are
reduced. The complex has also been shown to be present at centrosomes during early mitosis,
contributing to centrosome integrity by stabilizing the ch-Tog present there.
3,10
CHC17 also plays a role in endosomal membrane traffic during mitotic cell expansion and
abscission. CHC22 has also been implicated in this process, although in what capacity is still not
clearly understood.
1,11

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2.6 Gastric Acid Secretion and Parietal Cells
Secretion of digestive juices from stomach mucosa is a complex process involving multiple
transporters which ensure the optimum digestive conditions in stomach while still maintaining
the homeostasis of the secretory glands and stomach parietal cells. In the stomach, several types
of secretory epithelial cells make up highly organized tubular gastric glands. Of the three primary
types of secretory epithelial cells, parietal cells are responsible for secretion of the H
+
(hydronium
ion) from the cell cytoplasm to the gastric lumen (see Figure 8), and the primary transporter
involved in this process is called the H,K-ATPase.
Figure 8 Diagram showing histology of a gastric gland. Shown in the diagram are the chief cells, parietal
cells, mucous cells, gastric pits.

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2.7 H,K-ATPase
The H,K-ATPase is the transporter involved in gastric acid secretion. They are responsible
for secreting H
+
ions into the stomach and in exchange for the K
+
ions from the stomach lumen.
They can transport these ions against their large electrochemical gradients utilizing the energy
from the hydrolysis of ATP. The H,K-ATPase has two conformations, E1 and E2.
14,23,24

Figure 9 Proposed structure and enzymatic reaction of H,K-ATPase. (A) Overall structures and ion-binding
site architectures of H,K-ATPase. (B) Schematic illustration of P-type ATPase structures in different catalytic states
ATPc and ATPm designate catalytic and modulatory binding of ATP, respectively. Major movements of the
cytoplasmic N, P, and A domains (red, blue, and yellow, respectively) and the transmembrane domain (gray) are
indicated by arrows. Ligands are represented as follows: nucleotide, green; phosphate during transition, yellow; fully
transferred phosphate, white; ion1, black; ion2, brown; Mg2 , purple; K , dark blue. The phosphorylatable aspartate
residue is symbolized by a black rectangle and the TGES loop by an orange arc.
14,24

The E1 conformation binds to a phosphate from ATP and a hydronium ion changing its
conformation to E2 which allows the hydronium ion to exit from the cytoplasm of the cell to the
P a g e | 26

lumen. At this point the H,K-ATPase binds to a potassium ion and this cause E2 to revert to E1
releasing the phosphate and the potassium ion in cytoplasm (see Figure 9). This converts the ATP
to ADP and the ADP is released from the proton pump. Another ATP then binds to the E1 and the
cycle repeats.
23,24

Figure 10 (A) The pump–leak model for gastric TVs isolated from non-secreting animal. The vesicles are
depicted with an ATP-driven proton pump (P) that transports H
+
into the vesicles in exchange for K
+
. Additional paths
for the free diffusion (Jdiff, leak) of H
+
, K
+
, and Cl
-
are indicated. Since intravesicular K
+
is a key substrate of the pump,
JKdiff is critical to pump turnover and H
+
gradient. Likewise, an anion must accompany K
+
flux into the vesicle, thus
JCldiff is critical to the pump. In the resting vesicles K permeability is very low, thus no H
+
gradient. When a K
+

ionophore like valinomycin is added JKdiff increases and H
+
gradient becomes large. (B) Schematic model of
stimulation associated (SA) vesicles isolated from acid-secreting stomach. Although like (A), the system also includes
specific channel proteins, JCl and JK, for the transport of K
+
and Cl
+
, thus transport is no longer limited by artificial
ionophores and can create large H
+
gradients. (C) Proposed means for regulating H
+
pump activity in vivo. When the
pump is contained within the cell in TVs, low K
+
permeability limits ATP dominant protein, but there are several other
obvious apical membrane-associated-proteins, including Ac and Ez.
14,24
This process is modulated by several factors which tightly regulate the pH of stomach
lumen by positive and negative feedback mechanisms. On withdrawal of the stimulus the H, K-
ATPase undergoes internalization that contributes to stopping the secretion process. To
understand this regulation, we must understand the parietal cell morphology.
14
P a g e | 27

The most abundant protein in parietal cells is H,K-ATPase which is observed in abundance
in a system of intracellular tubules and vesicles, the so-called tubulovesicles in resting parietal
cells. Addition of secretagogues to resting parietal cells results in observable morphological
changes, growth of longer microvilli concomitant with disappearance of cytoplasmic
tubulovesicles.
22
The increase in surface area of maximally stimulated parietal cells is about 5-10
times that of resting cells. It has been proved by morphological evidence that the surface area is
conserved, and the increase in the apical membrane area is equivalent to the decreasing surface
area of the disappearing tubulovesicles (see Figure 10). Various membrane dynamics associated
with this stimulation in terms of membrane transporters is shown above.
14,23,24
2.8 Acid secretion associated membrane dynamics
In non-stimulated cells H,K-ATPase exists in vesicles called, tubulovesicles. On
homogenization of the parietal cell and subsequent membrane fractionation, the H,K-ATPase-
rich vesicles are usually found in relatively low density microsomal membrane fraction. On the
other hand cells which receive stimulation, on receiving the stimulatory signals traffic the H,K-
ATPase to the apical membrane. When stimulated cells are homogenized and H,K-ATPase-rich
membrane vesicles are isolated, they are found in the larger, more dense membrane fraction,
the “stimulation associated” (SA) vesicles. Thus the H,K-ATPase exists in two different types of
vesicles depending on the state of the cell. The SAs have selective, active K
+
and Cl
-
channels. SAs
have much more complex composition of proteins than TVs. It has been observed that the resting
cells have many more TVs than SAs and the stimulated cells are almost depleted of TVs but show
much higher number of SAs.
14
P a g e | 28

2.9 Inhibition of Acid secretion
Although inhibitory signals for acid secretion do exist the primary effecting mechanism
seems to be withdrawal of stimuli. The withdrawal of stimulus has been shown to lead to a
progressive resequestration of the expanded apical membrane back into cytoplasmic
compartment. The observed massive internalization results in the recovery of most of the TVs
which follows the sorting of different cargos. There is a consensus among the experts that the
primary proteins implicated in this process are the coat proteins, associated proteins, and
dynamin.
23
The coat proteins implicated in the internalization process are not clearly understood. We
have been able to show the presence of Clathrin Heavy Chain 22 (CHC22) in the parietal cells of
hog and rabbit gastric membranes.
The other proteins implicated in the process may be COPI and COPII. The cellular
membrane to be internalized has been observed to be associated with all four types of adaptor
proteins (AP 1-4)
1,25
. As we have previously discussed CHC22 associates with all of them except
perhaps AP2.
25
There is some evidence of CHC22 associating with AP2 (and CLCs along with it)
but it has not been conclusive. It has also been shown that the beta-subunit of H,K-ATPase is
necessary for its uptake. The substitution of alanine for the tyrosine in its cytoplasmic domain
apparently leads it to the continuous expression of the H,K-ATPase at the apical canalicular
membrane, and not in tubulovesicles. This is hypothesized to be caused by inhibition of
internalization of H,K ATPase. The tyrosine-based internalization motif at the cytoplasmic tail of
P a g e | 29

the β-subunit of H,K-ATPase may inhibited due to the A to Y substitution and its inability to
interact with AP-2.
25

CD63 also interacts with the H,K-ATPase β-subunit as shown by their co-
immunoprecipitation, and their coexpression in fibroblast cells changes the subcellular
distribution of the H,K-ATPase β-subunit from a predominantly cell-surface distribution to an
intracellular one, consistent with the role of CD63 in the endocytic process of the β-subunit.
26

CD63 is thus implicated in the reuptake of the proton pumps.
26

Dynamin is another protein implicated in the reuptake of H, K ATPase. The protein
associated with ‘pinching off’ of clathrin coated vesicles, is shown to be present much more
abundantly in parietal cells than other cells in the gastric mucosa. The need for continuous
formation of TVs, SAs, internalization of membrane, i.e. membrane recycling, may be the reason
for this phenomenon.
14
Another characteristic of parietal cells implicating the involvement of clathrin in H,K-
ATPase regulation is the exceptional actin cytoskeleton. The parietal cell actin cytoskeleton needs
to be exceptionally plastic in nature to accommodate the huge amount of stress put upon it
during not only the increase of surface area but also the decrease in the surface area.
Interestingly, CHC22 has been shown to help actin cytoskeleton integrity during endocytosis
1
.
There are also proteins like ezrin and Hip1r which are known to associate with or regulate
the clathrin mediated endocytosis that are involved in the process of regulation of H, K ATPase.
Hip1r is shown to interact with both clathrin and actin cytoskeleton.
14,24

P a g e | 30

Chapter 3: Materials and Methods
3.1 Sample preparation
Samples were prepared from stomachs of freshly euthanized rabbits. Hog gastric
mucosae were obtained by the tissue procurement company Sierra for Medical Science (Whittier,
CA). The protocol of Wolosin and Forte was followed to purify tubulovesicles. Briefly, the mucus
was carefully removed from the stomach by blunt scraping with glass slides before scraping off
more vigorously with either the glass slide (rabbit) or a metal spatula (hog) the gastric membrane
cells to separate them from the connective tissue and collecting them in a beaker. The volume of
the homogenization buffer containing 125 mM mannitol, 40 mM sucrose, 1 mM EDTA-Tris, 5 mM
PIPES-Tris (MSEP) equivalent to the 12 times the weight of the tissue was added to this beaker.
The tissue was cut to the smallest possible size using the scissors while continuously being
submerged in the MSEP. The sample tissue was then homogenized by passing it 15 times under
a Potter-Elvehjem homogenizer. This homogenized tissue prep was then given a preparatory spin
at 12,000 RPM x 10 min (10,000 x g x 10 min) in a preparatory centrifuge. The sediment was
discarded and the supernatant was centrifuged at 19,000 RPM for 2.5 hours (40,000 x g x 2.5 hr);
this pellet was labeled as P100. The supernatant was discarded and the sediment pellet was
resuspended in MSEP using a syringe. This resuspended pellet was then layered onto the
previously prepared sucrose density gradient (sucrose solutions of 27% and 32% (w/v) in MSEP
in ultracentrifuge tubes carefully poured to maintain the interface between the two solutions).
The gradients were then centrifuged at 35000 RPM on an SW41 Ti rotor overnight at 4° C. The
samples R27 and R32 represent the membrane fractions obtained above the 27% and 32%
P a g e | 31

sucrose gradient respectively, from rabbit. Analogous fractions from hog gastric mucosa were
labeled H27 and H32. The fraction obtained that sedimented through 32% sucrose, but settled
on top of the density gradient pellet was labelled Pe. Additional gradient concentrations greater
than 32%, such as 40% and 45% were sometimes utilized.
3.2 Antibodies
For the CHC22 specific western blots the antibodies from R&D systems for anti-human-
CHC22 sheep antibodies were purchased. BD Biosciences anti-human-clathrin mouse antibodies
were used as positive control. Secondary antibodies for the positive control were IR 680 goat
anti- mouse while for the sample western blotting the IR 680 donkey anti- goat antibodies were
used.
3.3 Human Skeletal Muscle Lysate
Human Skeletal Muscle Lysate was purchased from Abcam (product ab29330).
3.4 Western Blot
Western blotting was carried out using either 8% or10% SDS-PAGE gels. The gels were
loaded with 8 to 20 μg of sample protein depending on the sample concentrations and run at the
voltage as given in the standard protocol (80V till protein bands pass the loading gel and 170V till
the run is completed). The gels were then transferred onto cellulose membrane in the transfer
chambers overnight at constant 25V. Blocking was done by 5% milk in Tris-buffered saline-Tween
20 (TBS-T). TBS-T was also used for all the washings. 1% BSA was used for dilution of primary
P a g e | 32

antibody, at 1:2000 dilution. 5% milk was used as dilution medium for secondary antibody at
1:10,000 dilution. Visualization was done on the Odyssey LICOR scanner.
3.5 Coomassie blue staining
A concurrent gel with the western blot was run for Coomassie blue staining for each
western blot. The standard protocol for Coomassie blue staining was followed with overnight
incubation for the staining and variable destaining incubation timings. Destaining was carried out
till satisfactory background clarity was obtained. Gel preservation was done by drying out the
stained gel between two sheets of cellophane overnight.

P a g e | 33

Chapter 4: Results
4.1 Clathrin is present in gastric microsomal membrane samples
H,K-ATPase-rich gastric microsomal membranes were purified from either hog or rabbit
gastric mucosae by sucrose density gradient centrifugation of the microsomal pellet obtained by
differential centrifugation of gastric mucosal homogenates. The H,K-ATPase fractionates at the
27% (w/v) and 32% sucrose interfaces. Gastric microsomal membranes designated as “pellet”
are membranes that settle, but do not actually form a pellet, at the bottom of the 32% sucrose
barrier. Occasionally, 40% and 45% sucrose barriers were incorporated into the density
gradients.
Gastric microsomal membranes were run on Coomassie Blue stained gels to assess the
composition of each sample and analyzed for the presence and relative abundance of specific
proteins on Western blots.

P a g e | 34

Figure 11- The samples for the anti-CHC17 clathrin blot were hog brain CCV preparation
(lane 1), rabbit 32% sucrose density gradient fraction (R32) (lane 2), crude gastric microsomal
pellets from hog (P100) (lane 3). The sample size for clathrin blotting was 6 µg protein. The
primary antibody was an anti-clathrin (CHC17) mouse monoclonal antibody. A positive clathrin
signal was observed in hog brain CCVs and R32. No signal was observed for P100. The signal for
clathrin from brain CCVs may be lower compared to R32 because of degradation of the sample
(old sample and long-term storage) with time.
A concurrent gel for Coomassie blue staining was also run with P100, R32, hog brain CCV.
The sample size was 6 µg of protein.

(A) (B)
P a g e | 35

Figure 12- The samples for the anti-CHC17 clathrin blot were rabbit 40% sucrose density
gradient fraction (R40) and 45% sucrose density gradient fraction (R45). The sample size was 15
µg of protein. The primary antibody was an anti-clathrin (CHC17) mouse monoclonal antibody. A
positive clathrin signal was observed in both of the samples. The signal intensity was similar for
both the lanes indicating similar enrichment of clathrin in both R40 and R45.
A concurrent gel for Coomassie blue staining was also run with samples of R45 and R40,
from left to right, respectively, after the molecular weight (mw) ladder. The sample size was 15
µg of protein.

(B)
(A)
P a g e | 36

Figure 13- The samples for the anti-CHC22 clathrin blot were R27, R32, R40, and R45. The
sample size was 6 µg of protein each for R27, R49, and R45, but for R32, the sample size was 3.5
µg of protein. The primary antibody was anti-CHC22 sheep polyclonal antibodies. There was no
apparent signal obtained for any of the samples with the anti-CHC22 antibody.

P a g e | 37

Figure 14- Same membrane as in Fig. 12, reprobed for CHC17. The anti-CHC17 clathrin
monoclonal antibody was reactive for all four samples with R27 being the most enriched sample,
as it had the strongest signal. The signal intensity decreased for R32 showing lower enrichment,
although this lower signal could also be a function of less protein. It further decreased for R40
and R45 showing that they had the lowest enrichment of clathrin.
A concurrent gel for Coomassie blue staining was also run with R27, R32, R40, and R45

(A)
(B)
P a g e | 38

Figure 15- The samples used were from a standard human skeletal muscle lysate. Human
skeletal muscles are known to contain CHC22 for the regulation of GLUT4. If we were to get a
good response from human skeletal muscle lysate, we could have used this as a positive control
for the anti-CHC22 sheep antibody. The sample size was 20 µg of protein, and the primary
antibody was anti-CHC22 sheep antibody. However, no signal was obtained for CHC22. Thus,
the reactivity of this antibody is questionable.

P a g e | 39

Figure 16- The samples used were from a standard human skeletal muscle lysate again to
test if the samples react to our positive control anti-CHC17 clathrin monoclonal antibody. The
sample sizes were 60 & 120 µg of protein. The primary antibody was an anti-clathrin (CHC17)
mouse monoclonal antibody. Both of the sample sizes showed anti-CHC17 reactivity.
A concurrent gel for Coomassie blue staining was also run with standard human
skeletal muscle lysate with 15 µg and 30 µg of proteins from left to right, respectively, after the
molecular weight (mw) ladder.

(B)
(A)
P a g e | 40

Figure 17- The samples for the anti-CHC22 clathrin blot were R27 and R32. The sample
size was 15 µg of protein. The primary antibody was anti-CHC22 sheep polyclonal antibodies.
Both R27 and R32 showed a positive response to the new antibody. The signal was observed to
be stronger for the R32 lane. The new samples were concluded to have better enrichment in the
R32 fraction.

Figure 18- The samples for the anti-CHC22 clathrin blot were R27, R32 and sucrose density
gradient pellets (Pe). The sample size was 20 µg of protein run in duplicate for each sample. The
primary antibody was anti-CHC22 sheep polyclonal antibodies. No signal was obtained.

P a g e | 41

Figure 19- Same membrane as in Fig. 17, reprobed for CHC17. with anti-CHC17 clathrin
mouse monoclonal antibody. Strong signals were observed for all the lanes indicating that the
R27, R32, Pe all had significant quantities of clathrin. The signal was strongest for R32 showing
maximum enrichment.
A concurrent gel for Coomassie blue staining was also run with samples of R27, R32, and
Pe, from left to right, respectively (with a duplicate for each), after the mw ladder.

(B)
(A)
P a g e | 42

Figure 20- The samples for the anti-CHC22 clathrin blot were R27, R32 and sucrose density
gradient pellets (Pe) from left to right. The sample size was 20 µg of protein for each sample. The
primary antibody was anti-CHC22 sheep polyclonal antibodies. The signal was strongest for R32
showing maximum enrichment.

P a g e | 43

Chapter 5: Conclusion and Future Direction
Based on our primary hypothesis that CHC22 may play a role in the regulation of H,K-ATPase
we had to first show that CHC22 is in fact present in the gastric parietal cells. We have been able
to observe the presence of CHC22 in rabbit gastric membrane fractions. The signal is not as high
as that for CHC17 but the protein can be detected with high concentration of the antiCHC22
antibodies. Either CHC17 is more abundant the tubulovesicular fractions compared to CHC22, or
the CHC17 antibody is of higher reactivity. We are continuing to investigate the presence of
CHC22 in the fresh sample preparation and we have recently obtained a western blot in which
we can observe a distinct band for Clathrin heavy chain 22 with anti-CHC22 sheep antibodies (see
Figure 19).
We plan to continue investigating the presence of CHC22 in different sample preparations
from hog and rabbit. We are also trying to obtain new anti-CHC22 antibodies with higher affinity
and specificity than what we currently have. It has been difficult to obtain a signal even from
human skeletal muscle lysate which is known to have the highest concentration of CHC22 in
human body with the current R&D systems anti-CHC22 antibodies.
We also want to do immunofluorescence on the non-stimulated and stimulated parietal cells
using the older (R&D Systems) and the newer antibodies. We should see the differences in the
colocalization of not only the H,K-ATPase, but also of CHC22. The most convincing experiment
for our hypothesis will be if we can show the colocalization of CHC22 and H,K-ATPase at the cell
membrane after the removal of stimulus, or co-localization of CHC22 with the H,K-ATPase in
tubulovesicles.
P a g e | 44

Although the data remains far from convincing regarding the hypothesis we are encouraged
by the recent results. The most difficult aspect currently is obtaining anti-CHC22 antibodies that
are not only highly specific but also have a very high affinity. The lack of affinity of anti-CHC22
antibodies towards the target protein remains the biggest hurdle we have.

P a g e | 45

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Abstract (if available)

Abstract Clathrin is considered to be the prototype vesicle coat protein whose self-assembly mediates sorting of membrane cargo and recruitment of lipid modifiers to help deform the membrane into a budding vesicle. Detailed knowledge of the biochemistry, structure, and interacting proteins of the ubiquitous CHC17 isoform of clathrin has accumulated since its first observation, isolation, and characterization, almost 50 years ago, but the characterization of the role of the recently-discovered CHC22 isoform of clathrin is still in a nascent stage. Although CHC17 has well defined roles in multiple cellular processes, the most prominent documented function of CHC22 remains its modulation of GLUT4 glucose transporter. Similarly the modulation of trafficking of the gastric H,K-ATPase by vesicle coat proteins is not clearly understood. Here we investigate the hypothesis that CHC22 may be involved in the regulatory process of H,K-ATPase, due to the similarities in the GLUT4 and H,K-ATPase trafficking. Western blotting has shown that CHC22 is in fact present in the gastric parietal cells of hog and rabbit. There remain a number of unanswered questions that require further investigation.

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Asset Metadata

Creator Jamenis, Anuj Sunil (author)

Core Title CHC22 and parietal cells

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/24/2017

Defense Date 06/30/2017

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag CHC22,clathrin,clathrin heavy chain 22,OAI-PMH Harvest,parietal cells

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Okamoto, Curtis (committee chair), Hamm-Alvarez, Sarah (committee member), MacKay, J. Andrew (committee member)

Creator Email anuj.jamenis@gmail.com,jamenis@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-412068

Unique identifier UC11214589

Identifier etd-JamenisAnu-5616.pdf (filename),usctheses-c40-412068 (legacy record id)

Legacy Identifier etd-JamenisAnu-5616.pdf

Dmrecord 412068

Document Type Thesis

Format application/pdf (imt)

Rights Jamenis, Anuj Sunil

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA