Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were signifi
Trang 1CD38 is expressed as noncovalently associated homodimers
on the surface of murine B lymphocytes
Miguel E Moreno-Garcı´a1,2, Santiago Partida-Sa´nchez3, Julie Primack3, Adriana Sumoza-Toledo2,
He´le`ne Muller-Steffner4, Francis Schuber4, Norman Oppenheimer5, Frances E Lund3and
Leopoldo Santos-Argumedo2
1
Departamentos de Biologı´a Celular and2Biomedicina Molecular, CINVESTAV-IPN, Mexico;3Trudeau Institute, Saranac Lake, New York, USA;4Laboratoire de Chimie Bioorganique, UMR 7514 CNRS/ULP, Strasbourg-Illkirch, France;5Department of Pharmaceutical Chemistry, UCSF, San Francisco, USA
CD38 is a transmembrane glycoprotein that functions as
an ectoenzyme and as a receptor Based on the structural
similarity between CD38 and ADP-ribosyl cyclase from
Aplysia californica, it was hypothesized that CD38 is
expressed as a homodimer on the surface of cells Indeed,
CD38 dimers have been reported, however, the structural
requirements for their stabilization on the plasma
mem-brane are unknown We demonstrate that the majority of
CD38 is assembled as noncovalently associated
homo-dimers on the surface of B cells Analysis of CD38
mutants, expressed in Ba/F3 cells, revealed that truncation
of the cytoplasmic region or mutation of a single amino
acid within the a1-helix of CD38 decreased the stability
of the CD38 homodimers when solubilized in detergent Cells expressing the unstable CD38 homodimers had diminished expression of CD38 on the plasma membrane and the half-lives of these CD38 mutant proteins on the plasma membrane were significantly reduced Together, these results show that CD38 is expressed as noncova-lently associated homodimers on the surface of murine
B cells and suggest that appropriate assembly of CD38 homodimers may play an important role in stabilizing CD38 on the plasma membrane of B cells
Keywords: B lymphocytes; CD38; homodimer stability; NAD+glycohydrolase; protein structure
CD38 is a type II transmembrane ectoenzyme expressed by
many cell types [1–3] CD38 plays an important role in
calcium signaling as it catalyzes the production of several
calcium mobilizing metabolites including
adenosine(5¢)-diphospho(5)-b-D-ribose (ADP-Rib), cyclic
adeno-sine(5¢)diphospho(5)-b-D-ribose (cADP-Rib) and nicotinic
acid-adenine(5¢)diphosphate (NAADP+) [4,5] In addition
to its role as an enzyme, CD38 can also serve as a receptor
on the plasma membrane of leukocytes and lymphocytes
For example, incubation of B lymphocytes with agonistic
antibodies to CD38 induces calcium mobilization, protein
phosphorylation, proliferation, class switching, rescue from
cell death and induction of apoptosis [1,6–10] In order to
understand the dual receptor and enzyme properties of
CD38, a number of structure/function studies have been
performed These studies have been guided by analyses
of two CD38 homologues, the cytosolic Aplysia
califor-nica ADP-ribosyl cyclase [11,12] and the mammalian
GPI-anchored NAD+ glycohydrolase, CD157 [13,14]
Crystallographic and X-ray diffraction analyses of these two proteins indicated that both proteins form noncova-lently associated homodimers [15,16] Thus, it has been proposed that CD38 is also likely to be expressed as a homodimer on the plasma membrane and, in agreement with this hypothesis, initial reports showed that high molecular mass aggregates of CD38 are formed after incubation of human erythrocytes with NAD+or 2-mercapto-ethanol [17] In addition, it was reported that CD38 formed dimers and oligomers on the membrane of CD38 trans-fected HeLa cells [18] It is not clear, however, whether CD38 is always present in a dimeric form on the surface
of cells as many groups have reported finding only the monomeric form of CD38 [19–21] Furthermore, it remains
to be determined whether CD38 dimers are formed via covalent or noncovalent interactions between monomers For example, it has been suggested that two extracellular cysteines in CD38 (Cys119 and Cys201 in human CD38 or Cys123 and Cys205 in mouse CD38) could form interdi-sulfide bonds between CD38 monomers [22] In agreement with this hypothesis, studies carried out with porcine heart, rat lung and rat hepatocytes showed that under nonreduc-ing conditions CD38 forms dimers, while under reducnonreduc-ing conditions CD38 is present in a monomeric form [23–25]
On the other hand, Umar et al have shown that CD38 oligomers, expressed by retinoic acid stimulated HL60 cells, are covalently stabilized by transglutaminase, suggesting an alternate biochemical mechanism for the stabilization of covalent CD38 oligomers [26] As these previous results are difficult to reconcile with one another, it is still unclear
Correspondence to L Santos-Argumedo, Departamento de
Biomedicina Molecular, CINVESTAV-IPN, Av IPN #2508 Col.
Zacatenco, cp 07360, Me´xico D.F., Me´xico.
Fax: + 52 55 5747 7134, Tel.: + 52 55 5061 3323,
E-mail: lesantos@mail.cinvestav.mx
Abbreviations: BS 3 , Bis(sulfosuccinimidyl)suberate; NP-40,
Nonidet-P-40; IEF, isoelectric focusing.
(Received 17 October 2003, revised 22 December 2003,
accepted 20 January 2004)
Trang 2whether CD38 normally forms dimers, and if so, whether
stable CD38 dimer formation is dependent on covalent
bonds between monomers In this report, we show that
CD38 primarily forms homodimers on the plasma
mem-brane of B lymphocytes Furthermore, we demonstrate that
the stability of the CD38 homodimers is highly dependent on
the detergent used to solubilize the cells and is less dependent
on the formation of interdisulphide bonds between CD38
monomers, indicating that the CD38 homodimers in B cells
are likely to be stabilized via noncovalent monomer–
monomer interactions Finally, using Ba/F3 cells stably
transfected with a number of different CD38 mutants, we
identified two domains of the CD38 protein that confer
stability to the homodimeric form of CD38 Functional
analysis revealed that the half-life of these unstable CD38
homodimers on the plasma membrane was significantly less
than wild-type CD38 resulting in reduced plasma membrane
expression Thus, these results suggest that assembly of
CD38 homodimers may influence the stable expression of
CD38 on the plasma membrane of B cells
Materials and methods
Mice, B cell purification and cell lines
Splenic B lymphocytes were purified from 6 to 8-week-old
BALB/c, C3H/HeJ, NMRI, C57BL/6 or C57BL/6-Cd38–//–
[27] mice with magnetic beads coupled with antibodies to
B220 (Miltenyi Biotech, Auburn, CA, USA) All research
mice at CINVESTAV and Trudeau Institute were
eutha-nized by CO2narcosis in accordance with the
recommenda-tions of the Panel on Euthanasia of the AVMA and in
compliance with the CINVESTAV and Trudeau Institute
IACUC guidelines The IL-3 dependent murine pro-B cell
line, Ba/F3 (a generous gift from D Campana, St Jude
Children’s Hospital, Memphis, TN, USA) was cultured in
complete B cell media [21] supplemented with 10% (w/v)
WEHI-3 supernatant (containing IL-3)
Cell lysis, immunoprecipitation, Western blot and
isoelectric focusing
B cells were lysed with 10 mM Tris/HCl (pH 7.3), 2 mM
Na3VO4, 0.4 mM EDTA, 10 mM NaF, 1 mM
phenyl-methanesulfonyl fluoride, 2 lgÆmL)1aprotinin and
leupep-tin and 1% of one of the following detergents: Nonidet-P-40
(v/v) (NP-40), Triton X-100 (v/v) (Sigma), Chaps (w/v)
(Polysciences Inc., Warrington, PA, USA), deoxy-BigChap
(w/v) (Pierce, Rockford, IL, USA), or digitonin (w/v) (Wako
pure chemicals Ltd, Japan) Cell lysates were incubated
over-night at 4C with 5 lg of anti-mouse CD38 monoclonal
antibody (NIM-R5 [1]) or a nonspecific rat IgG2a (Zymed,
San Francisco, CA, USA) together with a 30 lL slurry of
protein G beads (Zymed) Complexes were boiled in
Laemmli buffer containing 2-mercaptoethanol or
dithiotre-itol (Sigma) at the concentration indicated in the text To
analyze the samples under nonreducing conditions, the
samples were suspended in the Laemmli buffer in the absence
of reducing agents and then heated at 50C for 3 min
Immunoprecipitated proteins (25 lL) were loaded into
10% polyacrylamide gels, electrophoresed and transferred
to nitrocellulose (Scleicher and Schuell, Dassel, Germany)
The membranes were blocked with 5% bovine serum albumin (BSA) (Research Organics, Del Mar, CA, USA) and incubated with rabbit polyclonal antibody against CD38 [28] overnight at 4C followed by an anti-rabbit– HRP (DAKO, Carpinteria, CA, USA) for 2 h at room temperature Proteins were developed using chemilumines-cence (Amersham Pharmacia Biotech, Buckingamshire, England)
The two-dimensional isoelectric focusing (IEF) analysis
of immunoprecipitated CD38 was performed as reported by O’Farrel [29]
Preparation of CD38 mutant cDNA constructs and Ba/F3 stable transfectants
Expression vectors containing the full length coding region of murine CD38 (CD38-WT-pME18S/neo) or the CD38 cytoplasmic region mutant (CD38-lATG-pME18S/neo) have been previously described [19,30] CD38-E150L and CD38-G68E were generated by PCR using the CD38-WT expression vector as a template and the primers below Restriction sites are underlined and the altered nucleotides that correspond to the replacement amino acid codons are indicated in lower case italics: CD38-E150L, primer 1: 5¢-(TACTTGGATCCAGGGAAAGATGTTCACCCTG ctGGACACCCTG)-3¢; CD38-E150L, primer 2: 5¢-(CC CTCTAGACCAGATCCTTCACGTATTAAGTCT ACACG)-3¢; CD38-G68E, primer 1: 5¢-(GACATCTTC CTCGagCGCTGCCTCATC)-3¢; CD38-G68E, primer 2: 5¢-(CCCTCTAGACCAGATCCTTCACGTATTAAGTC TACACG)-3¢; CD38-G68E, primer 3: 5¢-(GATGAGGC AGCGCTCGagGAAGATGTC)-3¢; CD38-G68E, primer 4: 5¢-(GGGGAATTCATGGCTAACTATGAATTTAGC CAG)-3¢
The E150L PCR product was digested with BamHI/XbaI and was used to replace the BamHI/XbaI fragment of CD38-WT in pME18S/neo The two PCR products for G68E were digested with XhoI, XbaI and EcoRI and cloned
by three way ligation into the EcoRI and XbaI sites of pME18S/neo The entire CD38 coding sequence was then sequenced in both directions to ascertain that the appropri-ate mutation was introduced and that no polymerase or cloning errors had occurred
Ba/F3 cells (5· 106) were electroporated as described previously [21] and were cultured in Ba/F3 media containing Geneticin (G418, Gibco-BRL, Grand Island, NY, USA)
at 500 lgÆmL)1 After 10 days, the surviving CD38+cells were single cell cloned into a 96-well plate using a FACSVantage-DIVA (Becton-Dickinson, San Jose, CA, USA) At least 20 independent clones from each transfec-tion were stained to determine CD38 expression levels and
at least five individual clones were picked to expand and analyze experimentally
Measurement of cyclase and glycohydrolase activity
in Ba/F3 transfectant cell homogenates Transfected Ba/F3 cells were washed with NaCl/Pi, pelleted, snap frozen and stored at )70 C The mem-brane fraction was obtained and resuspended in 1 mL potassium phosphate buffer (50 mM, pH 6.8) using a Dounce–Potter homogenizer (Wheaton Science Products,
Trang 3Milville, NJ, USA) Protein concentration was determined
with the BCAprotein assay (Pierce) The catalytic activity
of CD38 in Ba/F3 cell homogenates was determined
by HPLC using the radiolabeled substrates
[carbo-nyl-14C]NAD and [adenosine-U-14C]NAD+ as described
previously [30] To normalize the enzyme activity of the
cell homogenates from the various Ba/F3 transfectants,
the enzyme activity (Vmax) was multiplied by a correction
factor that compensated for the total protein per cell and
the amount of CD38 expressed per cell This correction
factor was obtained by dividing the amount of protein per
cell (1.54· 10)7mg) by the amount of CD38 expressed
on the membrane of each Ba/F3 cell (mean fluorescence
intensities) and is represented in arbitrary units of CD38
per mg of total protein The protein concentration per cell
was determined by lysing a known number of Ba/F3 cells
and determining protein concentration by Bradford
ana-lysis This was repeated multiple times and the number
represents the average amount of protein (in mgs) per cell
Crosslinking with BS3
B cells were washed, resuspended in 7 mL NaCl/Pi, and
560 lL of a 25 mMsolution of
bis(sulfosuccinimidyl)suber-ate (BS3, Pierce, Rockford, IL, USA) in 5 mMsodium citrate
buffer was added dropwise to the cell suspensions giving a
final concentration of 2 mMBS3 Cells were incubated for 1 h
at 4C with gentle shaking The reaction was stopped with
140 lL 1M Tris/HCl (pH 7.5) Cell suspensions were
washed with NaCl/Piand prepared for lysis
FACS analysis
To measure CD38 expression on Ba/F3 cells, 5· 105cells
were stained with anti-CD38 Ig (NIM-R5-FITC, dilution
1 : 500) (Southern Biotech, Birmingham, AL, USA) for
30 min at 4C The cells were analyzed by cytometry using
a FACSCalibur (Becton-Dickinson, San Jose, CA, USA)
Surface biotinylation of proteins
To analyze the stability of CD38 on the plasma membrane of
the different Ba/F3 mutants, labeling of the surface proteins
with the membrane impermeable reagent
sulfo-NSH-LC-biotin (Pierce) was performed as described [31] and following
the manufacturer instructions Briefly, cultured Ba/F3 cells
(1· 107) or splenic B cells (2· 108) were washed two times
with sterile NaCl/Piand resuspended in 3 mL of NaCl/Pi
containing 0.5 mgÆmL)1of sulfo-NHS-LC-biotin The cells
were incubated for 30 min at room temperature or on ice,
followed by three washings with NaCl/Pi The Ba/F3 clones
were then resuspended in complete Ba/F3 media and splenic
B cells were resuspended in supplemented RPMI media
containing 100 UÆmL)1of IL-4 The cells were cultured at
37C, and 2 · 106Ba/F3 cells or 5· 107splenic B cells were
harvested at 0, 2, 10, 20 and 30 h The cells were lysed with
0.5–1 mL of lysis buffer containing 1% (v/v) NP-40, and
CD38 was immunoprecipitated as described above
Immu-noprecipitated CD38 was analyzed by Western blot using
streptavidin-HRP (Sigma), and then the membrane was
stripped and reanalyzed with rabbit anti-CD38 Ig and
finally anti-rabbit Ig–HRP
Results
CD38 forms homodimers in murine splenic B cells
To address whether murine CD38 is expressed as a homodimer in splenic B cells, we purified these cells from CD38 expressing and CD38 deficient (CD38 KO) mouse strains and lysed them in buffer containing 1% (v/v) NP-40 CD38 was immunoprecipitated with an anti-mouse CD38 monoclonal antibody (NIM-R5), electrophoresed under reducing or nonreducing conditions and then analyzed by Western blot using a polyclonal rabbit anti-mouse CD38 Ig (Fig 1A) Under nonreducing conditions, no CD38 reactive proteins were detected in the immunoprecipitates from CD38 KO cells (Fig 1A, lane 1) In contrast, two distinct
Fig 1 CD38 forms 95 kDa homodimers in B lymphocytes (A)
B lymphocytes (5 · 10 7 ) were isolated from the indicated mouse strains, including CD38 deficient mice (CD38-KO [27]) The cells were lysed with 1% (v/v) NP-40 and CD38 was immunoprecipitated with monoclonal antibody to CD38, NIM-R5 The samples were prepared either in the absence (lanes 1–5) or presence of 5% (v/v) 2-mercapto-ethanol (lanes 6–10) and CD38 was detected by Western blot as des-cribed in Materials and methods The relative molecular mass markers are indicated on the left of each figure The nonspecific IgH band present in all of the reduced samples (including the CD38-KO sample)
is indicated with an asterisk (B) Immunoprecipitated CD38 from BALB/c B cell lysates was boiled in the presence of 2-mercaptoethanol (lanes 1–3) or dithiothreitol (lanes 4–6) at the concentrations shown
in the figure (C) CD38 was immunoprecipitated from splenic B cell lysates, resolved by 2D isoelectric focusing (IEF) and detected by Western blot IEF spots of monomeric and dimeric forms of CD38 are indicated by arrows b-ME, 2-mercaptoethanol; DTT, dithiothreitol.
Trang 4molecular mass forms of CD38 were observed in the
immunoprecipitates from CD38-expressing cells; a 42 and a
95 kDa protein (p42 and p95) (Fig 1A, lanes 2–5) The
42 kDa protein is the expected size of glycosylated
mono-meric CD38 [19] while the 95 kDa protein is the
approxi-mate size of a CD38 dimer When the samples were boiled
and reduced in 2-mercaptoethanol, we observed nonspecific
bands of 68 kDa (corresponding to the immunoglobulin
heavy chain present in B lymphocytes) and 200 kDa
(data not shown) in all immunoprecipitates, including the
sample from the CD38 KO mice (Fig 1A, lane 6) In
addition to observing the nonspecific bands, we still detected
the p42 and p95 forms of CD38 in the CD38-expressing cells
(Fig 1A, lanes 7–10) This indicates that p95 was partially,
although not fully, resistant to reduction by
2-mercapto-ethanol Interestingly, even addition of higher
concentra-tions of 2-mercaptoethanol or another reducing agent,
dithiotreitol, did not completely ablate the p95 form of
CD38 (Fig 1B)
To determine the structural composition of the p95 form
of CD38, we first ruled out the possibility that the p95 form
was composed of a CD38 monomer associated with the
immunoglobulin heavy chain from the precipitating
anti-CD38 Ig (data not shown) Next, we showed that the p95
form of CD38 was easily detected when iodoacetamide was
included in all of the buffers in order to block any reactive
free cysteines (data not shown) This ruled out the possibility
that p95 was formed during the lysis and
immunopreci-pitation process Finally, we compared p42 and p95 for their
pattern of isoelectric points by IEF and 2D polyacrylamide
gel electrophoresis For p42 we observed two dominant
isoelectric points of 7.7 and 7.2 and two minor points at 7.4
and 7.1 (Fig 1C) Analysis of p95 revealed isoelectric points
of 7.7, 7.2 and 7.1 (Fig 1C) These points were located at
similar positions to the corresponding points in the p42
monomeric form We did not detect a protein spot at 7.4 in
p95; however, this protein species only represented a minor
form even in the CD38 p42 monomer Taken together, the
data indicate that the p95 form of CD38 appears to
represent a homodimeric form of CD38 as it is recognized
by both monoclonal and polyclonal antibodies against
CD38, and has essentially identical IEF points as the p42
monomer form of CD38
CD38 homodimers are expressed on the surface
of splenic B cells and are destabilized when solubilized
with type B surfactants (steroid-based detergents)
To determine whether CD38 is normally expressed in the
homodimeric form on the plasma membrane of B cells, we
purified splenic B cells from normal and CD38 KO mice
and treated them for 1 h with a nonpermeable chemical
crosslinker, BS3, in order to stabilize the CD38 homodimers
during the lysis and immunoprecipitation steps As
expec-ted, no CD38 protein was detected in the CD38 deficient
cells (Fig 2A, lanes 2 and 5) Similarly to the previous
results, the majority of CD38 protein was of monomeric size
(p42) in the cells that were not treated with BS3(Fig 2A,
lanes 1 and 4) In contrast, in cells that had been treated
with crosslinker, CD38 was found predominantly in the
p95 homodimeric form (lanes 3 and 6) As the ratio of
homodimers to monomers was approximately five-fold
increased when the crosslinker was used (Fig 2A, compare lanes 1 and 3 or lanes 4 and 6), these results indicate that
a large proportion of the total CD38 is expressed in a
Fig 2 CD38 is found as homodimers on the surface of splenic
B lymphocytes and the stability of the dimers depends on the detergent used to solubilize the cells (A) Purified B cells were incubated with the nonpermeable crosslinker BS 3 for 1 h at 4 C (lanes 2, 3, 5 and 6) or left untreated (lanes 1 and 4) Crosslinked cells were lysed and CD38 was immunoprecipitated from cells expressing CD38 (CD38-WT, lanes 1, 3, 4 and 6) or lacking CD38 (CD38-KO, lanes 2 and 5) Immunoprecipitated proteins were treated with (lanes 4–6) or without 5% (v/v) 2-mercaptoethanol (lanes 1–3) and CD38 was detected by Western blot (B) Splenic B cells were solubilized with 1% (v/v) NP-40 (lanes 1, 2, 5 and 6) or Chaps (lanes 3, 4, 7 and 8) and CD38 was immunoprecipitated The samples were heated in the presence (lanes 5–8) or absence (lanes 1–4) of 5% (v/v) 2-mercaptoethanol and CD38 was detected by Western blot (C) B cells were solubilized with 1% (v/v) NP-40 (lane 1), Triton X-100 (lanes 2 and 3), digitonin (lanes 4 and 5), Chaps (lanes 6 and 7) or deoxy-BigChap (lanes 8 and 9) Lysates were immunoprecipitated with antibody to CD38 (NIM-R5) and CD38 was detected by Western blot The nonspecific IgH band present in all samples, including samples immunoprecipitated with an isotype control antibody (IgG2a), is indicated with an asterisk b-ME, 2-mercaptoethanol.
Trang 5homodimeric form on the surface of live B lymphocytes and
suggest that most of the CD38 dimers must fall apart when
the cells are solubilized in detergent Crosslinkers like
disuccinimidyl suberate, that have the same reactivity and
spacer arm length as BS3(11.4 A˚) also stabilized the CD38
homodimers However, crosslinkers such as
3,3¢-dithio-bis(sulfosuccinimidyl propionate), sulfo-disulfosuccinimidyl
tartarate and
sulfo-bis[2-(sulfosuccinimidooxycarbonyl-oxy)ethyl]sulfone, that have the same reactivity as BS3but
have different spacer arm lengths (12, 6.4 and 13 A˚,
respectively), were unable to stabilize the homodimers (data
not shown) These results suggest that the stabilization
of CD38 homodimers by crosslinkers depends strongly
on the conformation and orientation between the CD38
monomers
It has been reported that NP-40 and Triton X-100
stabilize noncovalent hetero- or homo-dimerization of
proteins, while detergents like Chaps and octylglucoside
disrupt these interactions [32,33] Up to now, in all our
experiments, the cells were solubilized in NP-40, a
deter-gent that might help to stabilize or protect the CD38
dimers from dissociating during the solubilization process
In sharp contrast, when the B cells were solubilized with
Chaps we found significantly less CD38 homodimers,
whether under reducing (Fig 2B, lanes 5–8) or
nonreduc-ing (Fig 2B, lanes 1–4) conditions This demonstrates that
the detergent used to solubilize the cells influenced the
amount of CD38 homodimers that could be
immuno-precipitated
To analyze whether the stabilization of CD38 dimers
was a property of the family of detergents utilized, we used
several different detergents to solubilize the cells As
shown in Fig 2C, CD38 dimers were precipitated when
the cells were solubilized with NP-40 or Triton X-100;
detergents that belong to the polyoxyethylene family
(Fig 2C, lanes 1–3) In contrast, when the cells were
solubilized with Chaps, digitonin or deoxy-BigChap,
members of the steroid-based detergent family, only
CD38 monomers were detected (Fig 2C, lanes 4–9)
These results suggest that CD38 homodimer stability is
dependent on noncovalent interactions between CD38
monomers
Structural requirements for CD38 homodimerization
To investigate the structural requirements for dimer stabilization, we determined whether different CD38 mutants were capable of forming homodimers when transfected into Ba/F3 cells Ba/F3 cells, stably transfected with full length wild-type CD38 (CD38-WT) or with different CD38 mutants, were solubilized in NP-40 lysis buffer and CD38 was immunoprecipitated, run on SDS/ PAGE under nonreducing conditions, and detected by Western blot Asummary of the results, presented in Table 1, indicates that CD38 homodimers were present in the lysates of most of the transfectants expressing CD38 mutants, including, CD38-E150L, a CD38 active site mutant (Table 2, [34]) and CD38-C123K, a mutant that
is unable to form the postulated interdisulphide bond between two CD38 monomers [22] These data indicate that CD38 homodimers can be formed even when the active site is altered and the putative interdisulphide bridge formed betweeen CD38 monomers is disrupted Interestingly, however, CD38 dimers were absent in lysates from two of the other mutant Ba/F3 transfectants
Table 1 Expression of CD38 homodimers in different CD38 mutants expressed in Ba/F3 pro-B cells Each of the mutant CD38 cDNAs listed, was stably expressed in Ba/F3 cells (Materials and methods) or A20 cells as described previously [30] The cells were solubilized in 1% (v/v) NP-40 CD38 was immunoprecipitated, run on SDS/PAGE gels under nonreducing conditions and then analyzed for the presence (Y)
or absence (N) of the p42 monomer and p95 homodimer by Western blot WT, wild-type.
Table 2 NAD+glycohydrolase activity of membrane homogenates from Ba/F3 transfectants Each of the mutant CD38 cDNAs listed, was stably expressed in Ba/F3 cells and the enzyme activity (V max ) of the membrane homogenates was determined as described previously [30] The V max was adjusted to reflect differences in CD38 expression levels between the various mutants and is reported as nmol of product formed per minute per arbitrary unit of CD38 Briefly, the total amount of protein per Ba/F3 cell was determined by the Bradford method The average amount of CD38 expressed on the membrane of each Ba/F3 cell was determined by FACS and is reported as mean fluorescence intensity (Fig 3C shows values of each of the clones) The relative enzyme activity of each of the mutants is given in parentheses It was determined by setting the V max adjusted activity of CD38-WT to 100% and then calculating the percentage activity of each of the mutants relative to CD38-WT MFI, mean fluorescence intensity; WT, wild-type.
Mutant
V max
(nmolÆmin)1Æmg)1protein)
Protein per cell (mg protein per cell · 10)7)
CD38 per cell arbitrary units CD38 per cell (1/MFI · 10)4)
V max adjusted (nmolÆmin)1per arbitrary units of CD38 · 10)10)
Trang 6In one of the mutants (CD38-lATG), the 22 amino acid
cytoplasmic region of CD38 was replaced with a 4 amino
acid tail (Met-Lys-Val-Lys), and in the second mutant
(CD38-G68E), the glycine at position 68 was replaced by
the polar residue glutamate The G68 residue is within the
a1-helix that has been previously postulated to be a dimer
interface site in the Aplysia enzyme [16] As shown in
Fig 3A(lanes 2 and 5), CD38 homodimers were
preci-pitated from Ba/F3 transfectants expressing CD38-WT or
expressing a mutant form of CD38 in which a single
residue in the active site was mutated (CD38-E150L) However, no CD38 homodimers were detected in immunoprecipitations from transfectants expressing the CD38-G68E or CD38-lATG mutant proteins (Fig 3A, lanes 3 and 4) This result suggests that the cytoplasmic region and first a-helix interface region of CD38 are important for dimer stability
To determine whether these two regions were necessary for CD38 dimer stabilization on the plasma membrane of the Ba/F3 cells, the transfectants expressing CD38-lATG
Fig 3 The stability and membrane expression of CD38 homodimers is dependent on at least two separate domains of CD38 (A) Ba/F3 cells transfected with control vector, CD38-WT, CD38-G68E, CD38-lATG or CD38-E150L were lysed with 1% (v/v) NP-40 and CD38 was imu-noprecipitated, run on SDS/PAGE under nonreducing conditions and detected by Western blot (B) The Ba/F3 transfectants listed above were treated (as described in Fig 2) with the crosslinker BS3(lanes 2, 4, 6, 8 and 10) or left untreated (lanes 1, 3, 5, 7 and 9) CD38 was detected by Western blot as in Fig 2A (C) Ba/F3 mutants were analyzed for expression of CD38 on the plasma membrane by FACS using the antibody, NIM-R5, conjugated to FITC Dead cells were excluded by propidium iodide incorporation Light line histograms, nontransfected Ba/F3 cells; dark line histograms, Ba/F3 transfectants The mean fluorescence intensity of the cells from each of the transfectants is listed above the histogram (D) Post-nuclear supernatants were prepared from Ba/F3 clones lysed with 1% (v/v) NP-40, the protein concentration was determined and equivalent amounts of protein were run on SDS/PAGE gels under reducing conditions CD38 and actin expression were analyzed by Western blot using rabbit polyconal anti-CD38 Ig and mouse monoclonal antibody to actin, followed by HRP-labeled anti-rabbit IgG and anti-mouse IgG, respectively (E) Comparison of plasma membrane and total CD38 expression levels in the Ba/F3 clones To determine the relative plasma membrane expression levels of CD38 between the different Ba/F3 clones the mean fluorescence intensity for each of the clones was determined (C) and the relative levels were normalized to that of the CD38-WT transfectant which was set at 100% To quantitate the total CD38 expression levels for the various Ba/F3 transfectant clones, densitometric analysis of CD38 and actin Western blots (D) were performed using SIGMAGEL LNK The CD38 levels for each clone were first normalized to actin by dividing the densitometric value of CD38 by the densitometric value of actin Then the relative total CD38 expression levels for each clone were normalized to CD38-WT which was set at 100%.
Trang 7or CD38-G68E were crosslinked with BS3, solubilized in
NP-40 lysis buffer, and CD38 was detected by
immuno-precipitation and Western blot (Fig 3B) As we have
previously observed, homodimers of CD38 were absent in
the immunoprecipitates from the noncrosslinked
CD38-lATG and CD38-G68E transfectants (lanes 5 and 9)
However, when the crosslinker was added, CD38
homo-dimers could be visualized (lanes 6 and 10) Indeed, similar
ratios of homodimers to monomers were observed in the
crosslinked CD38-G68E and CD38-lATG mutants
com-pared to crosslinked CD38-WT and CD38-E150L (compare
lanes 4, 6, 8 and 10) Therefore, the cytoplasmic region and
a1-helix domains are not critical for CD38 dimer
stabiliza-tion in B cells, however, the two domains must contribute to
the overall stability of CD38 homodimers because the
mutated dimers fell apart even under permissive
solubili-zation and nonreducing conditions
CD38-G68E and CD38-lATG are less efficiently
expressed and have a reduced half-life
on the plasma membrane
The previous results indicated that CD38-lATG and
CD38-G68E are not obligatory for dimer stabilization
but do contribute to the overall stability of the dimers,
particularly upon detergent solubilization It has been
reported that inappropriate folding of proteins or
inappro-priate assembly of multimeric protein complexes can
influence the surface and overall expression of these proteins
in cells and can also alter the half-life of the misfolded or
disorganized protein complexes [35,36] Given that the
stability of CD38-lATG and CD38-G68E homodimers is
reduced when the proteins are solubilized in permissive
detergents, immunoblotting and FACS experiments were
performed in order to analyze the total and surface levels of
CD38 in all the Ba/F3 transfectants (Fig 3C–E) These
experiments revealed that the total amount of CD38 (as
assessed by immunoblotting), as well as the amount of
CD38 expressed on the plasma membrane (as assessed by
FACS) was similar in the CD38-WT and CD38-E150L
transfected Ba/F3 cells (Fig 3C,D) In contrast, CD38
expression levels (both total and plasma membrane levels) in
transfectants expressing CD38-G68E and CD38-lATG
were significantly decreased relative to CD38-WT
(Fig 3C,D) Similar results were obtained upon analysis
of multiple independent Ba/F3 clones expressing
CD38-lATG or CD38-G68E (data not shown) Upon
densito-metric analysis of the immunoblots it became clear that the
amount of CD38 expressed on the plasma membrane and
the total amount of CD38 expressed by the various
transfectants correlated very well with one another
(Fig 3E), strongly suggesting that the reduced cell surface
expression of CD38 by the CD38-lATG and CD38-G68E
transfectants was not due simply to inefficient transport of
the protein to the plasma membrane In addition, confocal
microscopic analysis of CD38 expression in Ba/F3
trans-fectants revealed that neither CD38 nor any of the CD38
mutant proteins were present in intracellular compartments
(data not shown) Therefore, we next considered the
possibility that the reduced plasma membrane expression
of the CD38-lATG and CD38-G68E mutant proteins
could be due to a faster turnover rate for these mutant
molecules on the plasma membrane [35] To analyze this possibility, we performed pulse-chase experiments using normal B cells and the Ba/F3 transfectants To first determine whether the surface half-life of CD38 in Ba/F3 cells is comparable to that of splenic B cells, we biotinylated the surface of splenic B cells and Ba/F3 clones (CD38-WT and lATG) and compared the plasma membrane half-life
of CD38 In these experiments the biotinylation was carried out for 30 min on ice in order to avoid any potential internalization of biotin or biotinylated proteins The cells were then washed to remove the reactive biotin and cultured for up to 30 h The amount of cell surface biotin-labeled CD38 at various timepoints was determined by immuno-precipitating with anti-CD38 and immunoblotting with SA-HRP to detect the cell surface biotinylated-CD38 and anti-CD38 to detect the total CD38 pool The plasma membrane expression of CD38 on CD38-WT Ba/F3 transfectant cells and on normal B cells was quite stable over time with a half-life of approximately 28 h on both cell types (Fig 4A,B), indicating that the turnover rate of CD38
in both cell types is similar and comparable In contrast, the surface half-life of CD38-lA TG was less than half that observed for CD38 expressed by normal B cells or Ba/F3 transfectants, suggesting that this mutant protein is less stably expressed on the plasma membrane (Fig 4A,B) To confirm these results, we repeated the biotinylation experi-ment using Ba/F3 transfectants expressing other CD38 mutant proteins The plasma membrane expression of both CD38-WT and CD38-E150L was quite stable over time with a half-life of approximately 28 h (Fig 5A,B) In striking contrast, the half-lives of plasma membrane bound CD38-G68E and CD38-lATG were less than half that observed with CD38-WT (Fig 5A,B) Thus, both of the CD38 mutant proteins that form unstable homodimers also have significantly reduced stability on the plasma mem-brane, suggesting that appropriate assembly or stabilization
of CD38 into homodimers may be required for its extended expression on the plasma membrane
Enzyme activity is not dependent on the presence
of stable homodimers
As the mutations in the cytoplasmic region and the a1-helix
of CD38 affected dimer stability upon solubilization, plasma membrane expression levels and surface half-life, it was also possible that these mutations would affect the enzyme activity of the proteins To test the enzyme activity
of the CD38 mutants, membrane homogenates from the various Ba/F3 transfectants were prepared and NAD+ glycohydrolase activity in the membranes was measured by HPLC As shown in Table 2, the enzyme activity of the active site mutant, CD38-E150L was greatly decreased compared to CD38-WT Interestingly, the glycohydrolase activities of CD38-G68E and CD38-lATG were also less than CD38-WT As the membrane expression levels of CD38-lATG and CD38-G68E were reduced compared to CD38-WT (Fig 3C,D), we performed a calculation to adjust the enzyme activity (Vmax adjusted) to reflect the amount of total protein and CD38 protein expressed on
a per cell basis Upon adjusting the enzyme activity to compensate for the membrane CD38 expression levels, we found that the enzyme activity of CD38-lATG was at least
Trang 8as high as CD38-WT (Table 2) These data indicate that the
catalytic activity of CD38 is not dependent on the formation
of stable CD38 homodimers Interestingly, however, even
when expression levels of CD38-G68E were accounted for,
the NAD+glycohydrolase activity of CD38-G68E was only
27% of CD38-WT This result shows that a single point
mutation in the first a-helix of CD38, a residue that is far
removed from the active site of CD38, can significantly
influence CD38 enzyme activity
Discussion
In this work we show that homodimers of CD38 are expressed on the surface of B lymphocytes Although CD38 dimers that are sensitive to reducing agents have been previously reported [22–25], we found that the stability of CD38 dimers expressed in B cells correlated better with the type of detergent used to solubilize the cells (Fig 2) than the presence or absence of reducing agents (Fig 1) Previous reports have shown that heterodimerization of proteins such
as Bax with Bcl-2 or Bax with Bcl-XLare dependent on the detergent used to solubilize the cells [32,33] Thus, NP-40 and Triton X-100, detergents that form large micelles, stabilized the hydrophobic interactions between Bax and its partners, while Chaps and octyl glucoside, detergents that form small micelles, could not accommodate the hetero-dimers Interestingly, we found the same pattern with CD38 homodimers in that they were stabilized in the
polyoxy-Fig 5 The mutants expressing unstable CD38 homodimers present a reduced CD38 half-life on the plasma membrane (A) Ba/F3 transfectant cells expressing CD38-WT or each of the different mutants were sur-face labeled with sulfo-NHS-LS-biotin for 30 min at room tempera-ture The cells were washed and then cultured at 37 C for an additional 30 h 2 · 10 6
cells were harvested at 0, 2, 10, 20 and 30 h after biotin labeling Cell viability, as measured by trypan blue exclu-sion, was over 95% at each time point Immunoprecipitation and Western-blotting was performed as described in Materials and methods and Fig 4 (B) Densitometric analyses using the program
SIGMAGEL.LNK were performed to compare the relative amounts of biotin-labeled CD38 in each Ba/F3 clone Densitometry was per-formed as described in Fig 4.
Fig 4 The half-life of CD38 is the same in splenic B cells and
CD38-WT Ba/F3 transfectants Purified splenic B cells, CD38-CD38-WT and
CD38-lATG Ba/F3 transfectants were labeled with
sulfo-NHS-LS-biotin for 30 min on ice The cells were washed and then cultured at
37 C for an additional 30 h 2 · 10 6
Ba/F3 cells or 5 · 10 7
splenic
B cells were harvested at 0, 10, 20 and 30 h after biotin labeling Cell
viability, as measured by trypan blue exclusion, was over 95% for the
Ba/F3 transfectants at each time point and was 97, 90, 87 and 75% for
splenic B cells at 0, 10, 20 and 30 h after biotinylation, respectively.
(A) At each timepoint, the cells were lysed in 1% (v/v) NP-40, CD38
was immunoprecipitated with anti-CD38 Ig, and the
immunoprecipi-tated protein was analyzed by SDS/PAGE and Western blotting The
amount of plasma membrane associated (biotinylated-CD38) and
total CD38 was determined by immunoprecipitation, SDS/PAGE and
Western blotting Plasma membrane biotinylated-CD38 was detected
with streptavidin-HRP (left) Total immunoprecipitated CD38 was
detected using the polyclonal rabbit antibody to CD38 (right).
(B) Densitometric analyses using the program SIGMAGEL LNK were
performed to compare the relative amounts of biotin-labeled CD38
(membrane CD38) in each Ba/F3 clone To determine the relative
amount of biotin-labeled CD38 present in each clone, the
densito-metric value of biotin-CD38 was divided by the densitodensito-metric value of
total CD38 The ratio of cell surface CD38 to total CD38 at time 0
was set at 100% and all other time points were compared relative to
this.
Trang 9ethylene detergents (i.e NP-40 and Triton X-100) and were
destabilized with detergents such as digitonin, Chaps
and deoxy-BigChap (Fig 2) Importantly, these differences
could not be attributed to differences in the ability of the
various detergents to solubilize CD38 (data not shown)
Furthermore, when we used the crosslinker, BS3, the
majority of CD38 was captured in the homodimer form
indicating that CD38 must be dimerized via noncovalent
interactions that were partially disrupted when the cells were
solubilized in detergent However, it is also clear that
conformation and folding of the individual CD38
mono-mers is strongly influenced by the five known
intradisul-phide bonds present in each monomer and their reduction is
also expected to greatly influence the stability of the
noncovalently associated CD38 homodimers
As CD38 dimers appear to be stabilized via noncovalent
interactions between monomers, a reasonable assumption is
that mutations within the potential interface domains might
alter the formation or stability of CD38 homodimers When
cells expressing two different mutant forms of CD38, a
cytoplasmic region mutant and an a1-helix mutant, were
solubilized under nonreducing conditions in a permissive
detergent such as NP-40, we were unable to detect the
presence of CD38 homodimers (Fig 3A), suggesting that
these two domains play an important role in homodimer
stability Because homodimers of G68E and
CD38-lATG were observed when the cells were crosslinked with
BS3(Fig 3B), these data suggest that these domains are not
obligate for the stabilization of the dimers, but rather must
contribute to the overall stability of the dimers
Crystallo-graphic analysis of the Aplysia cyclase indicates four
putative oligomerization sites including the a1, a4 and a10
helices and residues between 242 and 248 [16] Thus, we
propose that multiple contact points contribute in an
additive or synergistic manner to CD38 homodimer
stabil-ization Although the mutants described here are not
sufficient, in themselves, to control CD38 homodimer
stabilization, the results clearly demonstrate a role for the
a1-helix and the cytoplasmic region in stabilizing the
solubilized homodimers
The mutations in the cytoplasmic tail and a1-helix of
CD38 not only affected the stability of the CD38
homo-dimer upon solubilization, but also significantly diminished
the expression of the CD38 homodimer on the plasma
membrane Indeed, we were never able to isolate
CD38-G68E expressing transfectants expressing levels of CD38
comparable to CD38-WT, despite screening more than 25
individual clones (data not shown) As a whole, these data
suggest that CD38-G68E and CD38-lATG mutants were
either inefficiently assembled and transported to the
mem-brane or were less stable on the surface Our pulse-chase
experiments (Figs 4 and 5) clearly showed that the plasma
membrane half-life of these CD38 mutants was significantly
less than CD38-WT, suggesting that extended plasma
membrane expression of CD38 may depend on the presence
of stable CD38 homodimers Although further experiments
will be needed to prove this hypothesis, similar results were
obtained analyzing mutants of the dipeptidylpeptidase IV
CD26 [36] The close correlation between surface expression
of CD38 and total expression of CD38 suggests that the
mutant forms of CD38 are not preferentially retained in
the intracellular compartments (Fig 3C–E) Furthermore,
intracellular expression analysis of CD38 on Ba/F3 trans-fectants and normal B cells using confocal microscopy or subcellular fractionation and immunobloting revealed that neither CD38 nor any of the CD38 mutant proteins analyzed in this study were detected in intracellular mem-branes (data not shown)
CD38 homodimers have been proposed to play a number
of different functional roles including formation of a transmembrane pore, allowing for transport of cADPR into the cytosol [37] However, it is clear that stable homodimers are not obligate for enzyme activity as the unstable CD38 homodimer mutant, CD38-lATG, had perfectly normal enzyme activity when the activity was adjusted to reflect CD38 expression levels on the membrane (Table 2) In agreement with this, we also observed NADase activity from the p42 monomeric form of CD38 (data not shown), suggesting that CD38 monomers are enzymatically active This is in agreement with the results of Bruzzone et al [18] Interestingly, although mutations in the active site do not decrease the formation or stability of CD38 homodimers (Table 2), the cells expressing the unstable CD38 homodimer, CD38-G68E, had significantly decreased CD38 dependent enzyme activity (Table 2) Thus, while CD38 enzyme activity is not critically dependent on the presence of stable CD38 homodimers, it
is clear that mutating a single residue in the a1-helix interface can decrease both homodimer stability and enzyme activity In conclusion, we have shown that CD38
is normally expressed as a noncovalently associated homodimer on the plasma membrane of B cells Mutations that affect the stability of the CD38 homodimer do not necessarily alter CD38-dependent enzyme activity; however, these mutations do result in reduced plasma membrane stability and decreased expression of CD38 on the plasma membrane
Acknowledgements The authors would like to thank Troy Randall for discussions and critical reading of this manuscript The authors also thank Dr Jose´ Manuel Herna´ndez-Herna´ndez for technical advice and Q F B He´ctor Romero Ramı´rez for technical assistance M E M.-G., A S.-T and L S.-A are supported by CONACyT Me´xico grants, # 28093N, 33497N and 40218Q J P., S P-S., and F E L are supported by NIH grant AI-43629 and the Trudeau Institute.
References
1 Santos-Argumedo, L., Teixeira, C., Preece, G., Kirkham, P.A & Parkhouse, R.M.E (1993) AB lymphocyte surface molecule mediating activation and protection from apoptosis via calcium channels J Immunol 15, 3119–3130.
2 Mehta, K., Shahid, U & Malavasi, F (1996) Human CD38, a cell-surface protein with multiple function FASEB J 10, 1408– 1417.
3 Lund, F.E., Cockayne, D.A., Randall, T.D., Solvason, N., Schuber, F & Howard, M.C (1998) CD38: a new paradigm in lymphocyte activation and signal transduction Immunol Rev.
161, 79–93.
4 Howard, M.C., Grimaldi, J.C., Bazan, J.F., Lund, F.E., Santos-Argumedo, L., Parkhouse, R.M.E., Walseth, T.F & Lee, H.C (1993) Formation and hydrolysis of cyclic ADP-ribose catalyzed
by lymphocyte antigen CD38 Science 262, 1056–1059.
Trang 105 Aarhus, R., Graeff, R.M., Dickey, D.M., Walseth, T.F & Lee,
H.C (1995) ADP-ribosyl cyclase and CD38 catalyze the synthesis
of a calcium-mobilizing metabolite from NADP + J Biol Chem.
270, 30327–30333.
6 Kirkham, P.A., Santos-Argumedo, L., Harnett, M.M &
Park-house, R.M.E (1994) Murine B-cell activation via CD38 and
protein tyrosine phosphorylation Immunology 83, 513–516.
7 Kitanaka, A., Suzuki, T., Ito, C., Nishigaki, H., Coustain-Smith,
E., Tanaka, T., Kubota, Y & Campana, D (1999)
CD38-medi-ated signaling events in murine pro-B cells expressing human
CD38 with or without its cytoplasmic domain J Immunol 162,
1952–1958.
8 Kumagai, M., Coustan-Smith, E., Murray, D.J., Silvennoinen, O.,
Murti, K.G., Evans, W.E., Malavasi, F & Campana, D (1995)
Ligation of CD38 suppresses human B lymphopoiesis J Exp.
Med 181, 1101–1110.
9 Zubiar, M., Guirado, M., Terhorst, C., Malavasi, F & Sancho, J.
(1999) The CD3-cde transducing module mediates CD38-induced
protein-tyrosine kinase and mitogen-activated protein kinase
activation in Jurkat T cells J Biol Chem 274, 20633–20642.
10 Yasue, T., Baba, M., Mori, S., Mizoguchi, C., Uehara, S &
Takatsu, K (1999) IgG1 production by sIgD+ splenic B cells and
peritoneal B-1 cells in response to IL-5 and CD38 ligation Int.
Immunol 11, 915–923.
11 Hellmich, M.R & Strumwasser, F (1991) Purification and
char-acterization of a molluscan egg-specific NADase, a
second-messenger enzyme Cell Regul 2, 193–202.
12 Lee, H.C & Aarhus, R (1991) ADP-ribosyl cyclase: an enzyme
that cyclizes NAD + into a calcium-mobilizing metabolite Cell
Regul 2, 203–209.
13 Dong, C., Wang, J., Neame, P & Cooper, M.D (1994) The
murine BP-3 gene encodes a relative of the CD38/NAD+
glyco-hydrolase family Int Immunol 6, 1353–1360.
14 Itoh, M., Ishihara, K., Tomizawa, H., Tanaka, H., Kobune, Y.,
Ishikawa, J., Kaisho, T & Hirano, T (1994) Molecular cloning of
murine BST-1 having homology with CD38 and Aplysia
ADP-ribosyl cyclase Biochem Biophys Res Commun 203, 1309–1317.
15 Yamamoto-Katayama, S., Ariyoshi, M., Ishihara, K., Hirano, T.,
Jingami, H & Morikawa, K (2002) Crystallographic studies on
human BST-1/CD157 with ADP-ribosyl cyclase and NAD
gly-cohydrolase activities J Mol Biol 316, 711–723.
16 Prasad, G.S., McRee, D.E., Stura, E.A., Levitt, D.G., Lee, H.C &
Stout, C.D (1996) Crystal structure of Aplysia ADP ribosyl
cyclase, a homologue of the bifunctional ectozyme CD38 Nat.
Struct Biol 3, 957–964.
17 Franco, L., Zocchi, E., Calder, L., Guida, L., Benatti, U &
De Flora, A (1994) Self-aggregation of the transmembrane
glycoprotein CD38 purified from human erythrocytes Biochem.
Biophys Res Commun 202, 1710–1715.
18 Bruzzone, S., Guida, L., Franco, L., Zocchi, E., Corte, G & De
Flora, A (1998) Dimeric and tetrameric forms of catalytically
active transmembrane CD38 in transfected HeLa cells FEBS Lett.
433, 275–278.
19 Harada, N., Santos-Argumedo, L., Chang, R., Grimaldi, J.C.,
Lund, F.E., Brannan, C.I., Copeland, N.G., Jenkins, N.A., Heath,
A.W & Parkhouse, R.M (1993) Expression cloning of a cDNA
encoding a novel murine B cell activation marker Homology to
human CD38 J Immunol 151, 3111–3118.
20 Jackson, D.G & Bell, J.I (1990) Isolation of a cDNAencoding
the human CD38 (T10) molecule, a cell surface glycoprotein with
an unusual discontinuous pattern of expression during
lympho-cyte differentiation J Immunol 144, 2811–2815.
21 Lund, F.E., Yu, N., Kim, K.M., Reth, M & Howard, M.C.
(1996) Signaling through CD38 augments B cell antigen
receptor (BCR) responses and is dependent on BCR expression.
J Immunol 157, 1455–1467.
22 Han, M.K., Kim, S.J., Park, Y.R., Shin, Y.M., Park, H.J., Park, K.J., Park, K.H., Kim, H.K., Jang, S.I., An, N.H & Kim, U.H (2002) Antidiabetic effect of a prodrug of cysteine, L -2-oxothiazolidine-4-carboxylic acid, through CD38 dimerization and internalization J Biol Chem 277, 5315–5321.
23 Chidambaram, N., Wong, E.T & Chang, C.F (1998) Differential oligomerization of membrane-bound CD38/ADP-ribosyl cyclase
in porcine heart microsomes Biochem Mol Biol Int 44, 1225– 1233.
24 Khoo, K.M & Chang, C.F (1998) Purification and character-ization of CD38/ADP-ribosyl cyclase from rat lung Biochem Mol Biol Int 44, 841–850.
25 Khoo, K.M & Chang, C.F (2000) Localization of plasma membrane CD38 is domain specific in rat hepatocyte Arch Bio-chem Biophys 373, 35–43.
26 Umar, S., Malavasi, F & Mehta, K (1996) Post-translational modification of CD38 protein into a high molecular weight form alters its catalytic properties J Biol Chem 271, 15922–15927.
27 Cockayne, D.A., Muchamuel, T., Grimaldi, J.C., Muller-Steffner, H., Randall, T.D., Lund, F.E., Murray, R., Schuber, F & Howard, M.C (1998) Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses Blood 92, 1324–1333.
28 Lund, F.E., Solvason, N.W., Cooke, M.P., Health, A.W., Grimaldi, J.C., Parkhouse, R.M., Goodnow, C.C & Howard, M.C (1995) Signaling through murine CD38 is impaired in anti-gen receptor-unresponsive B cells Eur J Immunol 25, 1338–1345.
29 O’Farrel, P.H (1975) High resolution Two-dimensional electro-phoresis of proteins J Biol Chem 250, 4007–4021.
30 Lund, F.E & Muller-Steffner, H.M., Yu, N., Stout, C.D., Schu-ber, F & Howard, M.C (1999) CD38 signaling in B lymphocytes
is controlled by its ectodomain but occurs independently of enzymatically generated ADP-ribose or cyclic ADP-ribose.
J Immunol 162, 2693–2702.
31 Cavet, M.E., Akhter, S., Murtazina, R., Sanchez de Medina, F., Tse, C.M & Donowitz, M (2001) Half-lives of plasma membrane
Na+/H+exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of degradation Am J Physiol Cell Physiol 281, C2039–C2048.
32 Hsu, Y.T & Youle, R.J (1997) Nonionic detergents induce dimerization among members of the Bcl-2 family J Biol Chem.
272, 13829–13834.
33 Hsu, Y.T & Youle, R.J (1998) Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations J Biol Chem 273, 10777–10783.
34 Munshi, C., A arhus, R., Graeff, R., Walseth, T.F., Levitt, D & Lee, H.C (2000) Identification of the enzymatic active site of CD38 by site-directed mutagenesis J Biol Chem 275, 21566– 21571.
35 Haardt, M., Benharouga, M., Lechardeur, D., Kartner, N & Lukacs, G.L (1999) C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis Anovel class of mutation J Biol Chem 274, 21873–21877.
36 Fan, H., Meng, W., Kilian, C., Grams, S & Reutter, W (1997) Domain-specific N-glycosylation of the membrane glycoprotein dipeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding Eur.
J Biochem 246, 243–251.
37 Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C & De Flora, A (1998) The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across mem-branes FASEB J 12, 1507–1520.