(Received for publication, August 24, 1995; and in revised form, December 5, 1995)
From the
Both apolipoprotein (apo) E and apoA-I are associated with lipoproteins, although with different particle classes. ApoE is associated with very low density lipoprotein (VLDL) and with the larger high density lipoprotein (HDL) subspecies, while apoA-I is found predominantly in association with most HDL subclasses. The genes encoding these proteins have a similar overall structure with the nucleotide sequences of the third and fourth exons coding for the mature protein. In an effort to understand the difference in lipoprotein association patterns of these two apoproteins, we have constructed and expressed chimeric apoproteins using cDNAs in which the third (n) and fourth (c) exons of human apoE and apoA-I are exchanged. McArdle rat hepatoma cells (McA-RH7777), which secrete VLDL- and HDL-like particles, were stably transfected with these cDNAs, and the cDNAs for human apoE and human apoA-I. Single spin NaBr gradient fractions of lipoprotein deficient serum-treated cell medium from transfected McA-RH7777 cells were analyzed. The distributions of transfected human apoE and apoA-I and endogenous rat apoE and apoA-I were compared with those of the chimeras. Among HDL subspecies, human apoE expressed by these cells is associated with particles of density 1.108 g/ml. Similarly, chimera apoA-InEc (exon 3 of apoA-I and exon 4 of apoE) is found in particles of density 1.111 g/ml. Human apoA-I, however, distributes in a broader range of particles with peak densities of 1.111 g/ml and 1.164 g/ml. The distribution of the complementary chimera, apoEnA-Ic, follows this same pattern, with peak particle densities of 1.098 and 1.137 g/ml. This is in contrast to the narrow distributions of endogenous rat apoE and apoA-I, which were found in particles of density 1.099 and 1.089 g/ml, respectively. When metabolically labeled medium was fractionated via gel filtration column chromatography, apoA-InEc was found to associate with the VLDL fractions; apoEnA-Ic was absent from these same fractions. These results suggest that the fourth exon largely determines the distinctive lipoprotein distribution patterns of these two human apoproteins and that the human apoA-I fourth exon sequence may account for the polydisperse HDL pattern as observed by others in transgenic mice expressing human apoA-I.
The soluble apolipoproteins appear to have evolved from a common
ancestral apoprotein, apoC-I, ()through whole-gene
duplications, intragenic duplications, and intragenic
deletions(1) . The set of current soluble apoproteins (apoA-I,
apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, and apoE) have maintained
similar overall gene structures. The first exon encodes the
5`-untranslated region, the second exon encodes most of the signal
peptide, the third exon encodes the rest of the signal peptide and the
amino terminus of the mature protein, and the fourth exon encodes the
remainder of the mature protein as well as the 3`-untranslated region.
In the case of apoA-IV, the first exon fulfills the role of exons one
and two in the other six apoproteins. In all cases, the last two exons
encode the mature plasma protein. The last exon of all the soluble
apoprotein genes codes for a variable number of 11 or 22-amino acid
repeats. These repeats are capable of forming amphipathic
helical
secondary structures that in vitro have been shown to be
important for binding to phospholipid vesicles (2) and
lipoproteins(3) . Despite the similar gene structure and the
presence of common secondary structure, these apoproteins all have
unique roles in lipoprotein metabolism and distinct lipoprotein
distribution patterns(4) .
Each apolipoprotein associates
with a specific class or classes of lipoprotein particles possessing
distinct compositional and size differences. It is this remarkable
specificity that determines the function and metabolic fate of each
class of lipoprotein particles. The present study focuses on apoE and
apoA-I, two apoproteins that associate with different classes of
lipoprotein particles. It is well known that apoE associates with VLDL
and the larger classes of HDL, such as HDL and
HDL
, whereas apoA-I associates predominantly with the
smaller HDL subspecies, HDL
and
HDL
(5) . We have constructed chimeric apoproteins
from these two human apoproteins in order to determine which domains
are responsible for their lipoprotein particle segregation patterns. We
examined the association of the apoproteins with nascent hepatic
lipoproteins secreted from the McA-RH7777 rat hepatoma cell line
transfected with expression vectors containing the chimeric cDNAs.
These cells are capable of vigorous triglyceride and apolipoprotein
production, and their apolipoproteins are secreted as lipidated
particles resembling VLDL and HDL of rat plasma(6) . Electron
microscopy reveals that these particles are primarily spherical in
shape(7) . Because this is a rat cell line, we were able to
differentiate between endogenous and transfected apoproteins using
species-specific antibodies. Therefore, we felt that these cells would
provide a good model in which to study the lipoprotein class targeting
of human apoE, human apoA-I, and chimeric proteins containing sequences
of each.
Gilbert (8) has suggested that the division of genes into exons separated by introns may correspond to coding regions for polypeptides with ``related or differing functions''(8) . Several well known proteins exhibit this kind of gene organization such as the LDL receptor(9) , immunoglobulin genes(10) , and the chick pyruvate kinase gene(11) . This evidence makes our chimeric approach a logical one. By exchanging homologous exonic regions between structurally related but functionally different apoproteins, we hoped to begin to identify those regions of apoE and apoA-I responsible for their specific lipoprotein class distribution. Chimeric apoproteins were expressed by creating cDNAs in which the corresponding third and fourth exon regions of human apoE and apoA-I were exchanged. This approach allows the isolation and study of a specific apoprotein domain in the context of an overall apoprotein structure.
Figure 1: A, construction of the pCMV4-EnA-Ic expression vector; B, construction of the pCMV4-A-InEc expression vector. The chimeric cDNAs were constructed using standard ligation and mutagenesis protocols as described under ``Experimental Procedures.'' ApoE sequences are shown in black. ApoA-I sequences are shown in stripes.
Next, the apoE signal peptide region and third exon were isolated
from the cDNA plasmid pHEBB6, which contains the BalI-HinfI fragment of the human apoE cDNA pE386
(obtained from J. Breslow, Rockefeller University, New York) subcloned
into the BamHI site of pUC19. The plasmid was digested with SacII (nucleotide 469), producing a 3` overhang that was
blunted with mung bean nuclease. BamHI linkers were added, and
the plasmid was digested with BamHI. The 428-base pair
fragment containing the apoE signal peptide and N terminus was
subcloned into the BamHI site of the pUC19-A-Ic plasmid. The
resulting plasmid, pUC19-EnA-Ic parent, was selected for proper
orientation of the apoE fragment, and is shown in Fig. 1A. In order to remove the nucleotides in
pUC19-EnA-Ic parent between the codons for arginine 61 in apoE and
leucine 44 in apoA-I, the 1100-base pair HindIII-EcoRI fragment in pUC19-EnA-Ic was subcloned
into the m13mp18 polylinker region at the corresponding sites. All 268
nucleotides between the codon for arginine 61 of apoE and the codon for
leucine 44 of apoA-I were deleted by oligonucleotide site-directed
mutagenesis in order to bring the two exons side by side and in frame.
Oligonucleotide site-directed mutagenesis was performed using the T-7
Gen in vitro mutagenesis system, based on the work by
Vandeyar(14) . Successful mutagenesis was determined by
analytical restriction endonuclease digestions, and dideoxynucleotide
sequencing of this central region using S-dATP
(>600Ci/mmol) and an oligonucleotide corresponding to apoprotein
sequences 40-60 nucleotides upstream from the site of
mutagenesis.
The final 890-base pair cDNA chimeric was subcloned into the HindIII-XbaI site of pCMV4(15) , downstream of the human cytomegalovirus (CMV) promoter and a translational enhancer from the alfalfa virus 4 RNA.
In an effort to understand the difference in lipoprotein association patterns of apoE and apoA-I, we have constructed and expressed chimeric apoproteins using cDNAs in which the corresponding third and fourth exon regions of human apoE and human apoA-I are exchanged. These two exons code for the amino acids found in the mature protein. We refer to the amino acids encoded by the third exon as the N terminus (designated as n) and the amino acids encoded by the fourth exon as the C terminus (designated as c). Chimera apoEnA-Ic is encoded by a construct that specifies the apoE signal peptide and residues 1-61 followed by apoA-I residues 44-243. The complementary chimera, apoA-InEc, contains the apoA-I signal peptide, propeptide, and residues 1-43 followed by apoE residues 62-299. This chimera was constructed with the E3 allele, i.e. containing a cysteine at position 112. It is not known whether McA-RH7777 secrete apoA-I as a proprotein or a mature apoprotein; however, experiments with HepG2 cells show that apoA-I is secreted into the culture medium as a proprotein(22) . We included the propeptide in the apoA-InEc chimera for the sake of experimental consistency, assuming that whether or not the propeptide is removed in our cell system, the wild-type human apoA-I and apoA-InEc transfectants would be processed in the same manner. McA-RH7777 cells are derived from a rat hepatoma cell line and secrete VLDL- and HDL-like particles(6) . We prepared individual stable transfectants of this cell line expressing human wild-type apoE and apoA-I and the chimeric apoproteins.
Figure 2:
Expression of chimeric apoproteins by
McA-RH7777 cells. McA-RH7777 cells transfected with pSV2-neo,
pCMV4-EnA-Ic or pCMV4-A-InEc were seeded at a density of 1.5
10
cells/T25 flasks and pulsed with 3 ml of 50 µCi/ml
Tran
S-label in DMEM/high glucose, 10% LPDS with 40
µM methionine for 24 h. Equal trichloroacetic acid
precipitable counts from each medium were immunoprecipitated
simultaneously with species-specific antibodies to rat apoE, rat
apoA-I, and human apoE. The immunoprecipitates were analyzed on
SDS-polyacrylamide gels, followed by fluorography as described under
``Experimental Procedures.'' Lane 1, neo-transfected
cells; lane 2, apoEnA-Ic-transfected cells; lane 3,
apoA-InEc transfected cells.
Figure 3:
Distribution of apoproteins on nascent
lipoproteins separated by equilibrium NaBr density gradients.
McA-RH7777 cells transfected with pSV2-neo or the human apoprotein
constructs were seeded at 1.5 10
cells/T25 flask.
After 24 h, the medium was replaced with 3 ml of DMEM/high glucose plus
10% LPDS for an additional 24 h. Two ml of medium was fractionated on a
NaBr density gradient as described under ``Experimental
Procedures.'' 120 µl of each fraction was subject to Western
blotting using anti-rat or human specific antibodies to apoE and apoA-I
and
I-protein A. After exposure to x-ray film, the
apoprotein bands were excised and counted, and cpms from each fraction
were graphed. The density of each fraction was determined by
refractometry. Panel A, single representative distribution
profile of rat apoE; panel B, single representative
distribution profile of rat apoA-I; panel C, composite
distribution profile of human apoE, n = 5; panel
D, composite distribution profile of human apoA-I, n = 4; panel E, composite distribution profile of
apoA-InEc, n = 2; panel F, composite
distribution profile of apoEnA-Ic, n =
4.
The association of the human apoE and apoA-I with nascent HDL particles separated by gradient centrifugation is shown in Fig. 3, C and D, respectively. The majority of both of these apoproteins is found associated with the nascent HDL particles. Like rat apoE, human apoE is found on lipoprotein particles that distribute as a single peak within the HDL density range of the gradient. The peak density is 1.108 g/ml, similar to the peak density of the endogenous rat apoproteins. However, the human apoE distribution is broader than that of either rat apoprotein E and A-I, and the broadening of this peak is toward the lighter and presumably larger lipoprotein fractions. In contrast to rat apoA-I and rat and human apoE, human apoA-I has a broader distribution and associates with lipoproteins of two distinct peak densities. The first peak has a density of 1.111 g/ml, and the second peak has a density of 1.164 g/ml. The broadness of the peaks is probably due to the high expression of human apoA-I. This polydisperse distribution pattern is characteristic of human apoA-I in the human HDL subclasses.
The association of the
chimeric apoproteins with nascent hepatic lipoproteins secreted from
the McA-RH7777 cells is shown in Fig. 3, E and F. ApoA-InEc is found on particles that distribute as a single
peak with a peak density of 1.111 g/ml. Very little of this chimeric
apoprotein was found in the lipid-poor ``bottom fractions.''
This distribution pattern is very similar to that of wild-type human
apoE (Fig. 3C). Similar to the results with human
apoA-I, apoEnA-Ic (Fig. 3F) was found associated with
two discrete particles within the HDL density range of these gradients.
The peak density of the lighter particles is 1.098 g/ml, similar to the
peak density of particles containing apoA-InEc. The peak density of the
denser particles is 1.137 g/ml. These densities correspond
approximately to the densities of human HDL and
HDL
, respectively. As with the apoA-InEc chimera, very
little of this chimeric apoprotein was found in the lipid-poor bottom
fractions.
To quantitate the information from these graphs, the area
under the curve, defined by the conventional HDL and
HDL
density ranges, was measured using the Sigma SCAN
computer program. Areas between d 1.063-1.125 g/ml and d 1.125-1.210 g/ml were expressed as a percentage of
total area described by the specific HDL apoprotein distribution
obtained from the medium of each stable cell line. As shown in Table 2, the percentage of the total apoEnA-Ic and human apoA-I
found in the HDL
and HDL
density ranges is
similar, with these apoproteins being distributed approximately equally
in these fractions. By contrast, the major proportion of apoA-InEc and
human apoE is found on particles in the HDL
density range.
Figure 4:
Nondenaturing gradient gels of
[P]orthophosphate-labeled lipoproteins. Cells
were seeded at a density of 0.75
10
/T25 flask. The
following day, the cells were incubated with a 5-ml pulse medium
containing DMEM/high glucose, 10% horse serum, 10% fetal calf serum, 1%
each penicillin, streptomycin, glutamine, 37.5 mg/liter
Na
HPO
7H
O, and 20 µCi/ml
[
P]orthophosphate. After 24 h, the cells were
chased with DMEM/high glucose plus 10% LPDS for an additional 24 h. 2
ml of each medium sample was run on 10-20% NaBr gradients as
described under ``Experimental Procedures.'' Peak fractions,
as determined by Western blotting for each cell line, were loaded onto
a 4-30% nondenaturing gradient gel. Lanes 1 and 2 are aliquots of 1.098 and 1.146 g/ml density fractions from human
apoA-I transfected cells, respectively; lanes 3 and 4 are aliquots of 1.098 and 1.136 g/ml density fractions from
apoEnA-Ic transfected cells, respectively; lane 5, the 1.098
g/ml density fraction from human apoE transfected cells; lane
6, the 1.098 g/ml fraction from apoA-InEc transfected cells; lane 7, the 1.098 g/ml density fraction from neo-transfected
cells.
To confirm that the P-labeled lipoproteins contain the
human wild-type and chimeric apoproteins, unfractionated media from the
stable transfected cells were analyzed on nondenaturing gradient gels,
which were subsequently immunoblotted with species-specific antibodies (Fig. 5). Human apoA-I and apoEnA-Ic are both found on two
distinct sized particles. ApoA-I is found on particles within the broad
range of 5.90-4.50 nm in radii and in particles with radii of
3.90 nm. ApoEnA-Ic is found on particles with radii of 5.80-4.75
nm and 4.00 nm. Human apoE was detected on particles with radii ranging
from 8.60 to 5.70 nm and a minor band at 4.3 nm. ApoA-InEc has a
similar pattern with this apoprotein detected on particles with radii
ranging from 8.50 to 5.15 nm and a minor band at 4.1 nm.
Figure 5:
Western blotting of unfractionated media
run on 4-30% nondenaturing gradient gels. McA-RH7777 cell lines
expressing human apoproteins, and chimeras were seeded at a density of
1.5 10
cells/T25 flask. After 24 h, the medium was
replaced with DMEM/high glucose with 10% LPDS for an additional 24 h.
60 µl of medium from each cell line was then loaded onto
4-30% nondenaturing gradient gels, transferred to Immobilon P
membrane, and probed with human specific antibodies, followed by ECL
detection. Comparison of the chimeric apoprotein lipoprotein
distributions with that of the wild-type human apoproteins is shown. Lane 1, human apoA-I medium; lane 2, apoEnA-Ic
medium; lane 3, human apoE medium; lane 4, apoA-InEc
medium. Note, lanes 1 and 2 were probed with
anti-human specific apoA-I antibodies, and lanes 3 and 4 were probed with anti-human specific apoE antibodies. The
following standards were used to generate a standard curve to calculate
the following radii: thyroglobulin, 8.5 nm; ferritin, 6.1 nm; catalase,
4.6 nm; lactate dehydrogenase, 4.1 nm; albumin, 3.55
nm.
Hepatoma cell medium
containing chimeric proteins labeled with TranS-label was
fractionated on these columns, and the VLDL peak was identified by
counting aliquots of fractions between 52 and 70. Equal amounts of
radioactivity from total media and the three VLDL peak fractions
isolated from the media of the two chimeric apoprotein expressing cells
were immunoprecipitated to ascertain the presence of the chimeric
apoproteins, rat apoE and rat apoA-I. As seen in Fig. 6A, apoA-InEc was associated with the nascent VLDL
particles (lanes 6-8), while apoEnA-Ic (lanes
2-4) was barely detectable. The low level of association of
apoEnA-Ic was not due to the absence of the protein in the media, since
approximately comparable levels of the two chimeric apoproteins were
immunoprecipitated from the unfractionated media (lanes 1 and 5). Wild-type human apoE is also associated with VLDL
particles (Fig. 6B). Rat apoE is present in the VLDL
fractions but at significantly lower levels in the presence of
apoA-InEc than apoEnA-Ic. This may be due to displacement of the rat
apoE from the VLDL particles by the apoE portion of the apoA-InEc
chimera. Negligible levels of rat apoA-I (Fig. 6A) and
human apoA-I (data not shown) are detected in the VLDL fractions as
expected.
Figure 6:
Association of wild-type and chimeric
apoprotein with VLDL particles. Cells were seeded and labeled with
TranS-label as described under ``Experimental
Procedures.'' 5 ml of medium was fractionated on Biogel A-5m
columns, and the VLDL peak was identified by counting an aliquot of
each fraction. Equal levels of radioactive media or fractions were
immunoprecipitated with anti-rat apoE antibodies, anti-rat apoA-I
antibodies, and anti-human apoA-I, simultaneously. A, lane
1, unfractionated medium from apoEnA-Ic-expressing cells; lanes 2-4, peak three VLDL fractions from
apoEnA-Ic-expressing cells; lane 5, unfractionated medium from
apoA-InEc-expressing cells; lanes 6-8, peak VLDL
fractions from apoA-InEc-expressing cells. B, samples from
human apoE expressing McA-RH7777 cells: total medium and combined peak
fractions 55/56, 57/58, 59/60, immunoprecipitated separately for human
apoE and rat apoE.
In this study, we have set out to determine whether the
domains of human apoproteins E and A-I encoded by the third or fourth
exons account for their selective targeting to VLDL and subsets of HDL.
Our results indicate that the selective targeting of apoproteins E and
A-I to nascent lipoproteins secreted from hepatoma cells is
attributable largely to the domains of these apoproteins encoded by the
fourth exons, which specify a series of amphipathic helices.
Preliminary studies utilizing the same chimeric proteins as used in
this study produced in Escherichia coli indicate that similar
domains function to target the apoproteins to mature human plasma
lipoproteins. However, we cannot discount a small contribution from
domains encoded by the third exons, although any effects these domains
have must be quite subtle. Our results also suggest quite strongly that
the domains encoded by the fourth exon of the apoA-I gene account for
the observed differences in the HDL subclass present in humans and
rodents.
The majority of the human apoE associated with HDL-like particles with a density similar to those particles secreted by the neo transfected cells, as observed on equilibrium density gradients, although there does appear to be a broader distribution of particles on the HDL population containing human apoE than those containing rat apoE. This broadening affects both a subpopulation of higher density particles that also contain human apoE as well as a set of lighter subfractions (fractions of density 1.07 and 1.08 g/ml, Fig. 3C). Thus, human apoE is capable of associating with particles of similar density as the rat apoproteins. However, when the sizes of the particles were examined on nondenaturing gels, the majority of the human apoE containing particles were larger (radii = 8.85-5.45 nm) than the HDL particles secreted from the neo-transfected cells (radius = 4.50 nm). The density of the particles with which apoA-InEc associates is again similar to that of human apoE with a monodisperse distribution, although not quite as diffuse as the latter. Thus it appears that the fourth exon encoded amino acids of human apoE allow the formation of these larger HDL particles without significantly altering the density of the particles.
In contrast to the distribution of rat apoA-I, human apoA-I distributes onto particles with two distinct densities and sizes. The particles in the first peak of the NaBr density gradients have the same density and size as those in the single peak of the control cells, while the particles in the second peak are more dense and smaller in size. ApoEnA-Ic, which contains the fourth exon of apoA-I, also associates with two different particles of distinct size and density. These results suggest that the fourth exon-encoded amino acids of apoA-I are sufficient to determine HDL association and the formation of HDL subclasses. In addition, they strongly suggest that amino acid differences in the fourth exon-encoded amino acids of rat and human apoA-I may account for the different HDL subclass formation in humans and rodents.
The different lipoprotein distribution patterns seen for rat apoA-I compared with human apoA-I, and as shown here apoEnA-Ic, have been observed previously in mice expressing human apoA-I as a transgene (23) . In control mice, the HDL profile was monodisperse, and the mouse apoA-I associated with particles 4.6 nm in radius. By contrast, the transgenic mice exhibited a biphasic HDL profile and the human apoA-I appeared in particles with radii of 5.1 and 4.15 nm. An explanation for these differences may reside in the primary sequence of the fourth exon-encoded amino acids. Since rat and mouse both appear to have monodisperse HDL distributions, while human HDL distributions are polydisperse (biphasic), the important contribution to the difference between rat and human must be shared between rat and mouse. There are 56 positions in the fourth exon in which both rat and mouse sequences differ from the human (Fig. 7). At 12 of these positions, there is either a proline or a charge change, and at five a change in hydrophobicity. The charge changes are clustered in helices 6 and 7, both of which are class A helices(24, 25) . The single proline change is at position +187 (using human sequence numbering) and may be the best candidate.
Figure 7: Fourth exon encoded amino acid sequence comparison of human, rat, and mouse apoA-I. The fourth exon-encoded sequences are separated into groupings of 11- and 22-amino acid helical repeats, which are separated by prolines. Rat and mouse amino acid residues that are different from human apoA-I are shown in lower case. Those not shown are identical to human residues. Boldface and underlined residues with &cjs1229; above represent charge changes or proline changes between human apoA-I and both rat and mouse apoA-I. Hydrophobic changes are marked with * above. Dash indicates a deletion.
Studies by Leroy et al.(26) have
suggested that differences in the helical repeat regions of apoA-I,
apoE, and apoA-IV may account for the spontaneous formation of discrete
sized reconstituted HDL (rHDL) particles in the presence of varying
amounts of palmitoyloleoylphosphatidylcholine. Generally, it was
observed that the apoprotein with the greatest number of repeats,
apoA-IV, was capable of forming the largest rHDL, while apoE formed
larger rHDL than did apoA-I. This phenomenon may explain the formation
of larger particles by the McA-RH7777 cells in the presence of human
apoE. Human apoE is found on the cholesterol ester-enriched classes of
HDL,
HDL/HDL
(18, 27, 28) ,
and its ability to conform to these larger particles, relative to
apoA-I, may play an important role in reverse cholesterol transport.
In our cell culture model, human apoE and apoA-InEc were also found
associated with the VLDL-like particles secreted by the McA-RH7777
cells. By contrast, neither human apoA-I nor apoEnA-Ic associated with
VLDL particles. Thus the third exon of each of these parent apoprotein
genes does not encode a strong VLDL targeting domain. In addition, the
rat apoE levels in the peak fractions from apoA-InEc transfected cells
appeared to be lower than in the apoEnA-Ic transfected cell line. This
difference suggests that apoA-InEc and rat apoE may be competing for
association with the VLDL-sized particles. However, the wild-type human
apoE does not appear to displace rat apoE despite being
expressed at higher levels than apoA-InEc. There may be a subtle effect
of the apoA-I third exon region in the apoA-InEc chimera, which needs
to be further explored before a full explanation can be reached.
In conclusion, we have established a model for efficient expression of chimeric apoproteins and have shown that the chimeric nature of these apoproteins is not detrimental to their expression or secretion. Our results support the conclusion that the fourth exon-encoded amino acids of apoE contain sequences that target apoE to VLDL and larger HDL classes. The fourth exons of both apoE and apoA-I also encode amino acid sequences that determine not only the association of each apoprotein with HDL but also the ability to form different subclasses of HDL particles. Thus it appears that it is the nature of the apoprotein secreted that is primarily responsible for the size and density of particles produced by hepatoma cells. It remains to be established whether sequences encoded by exon 4 alone are sufficient to determine lipoprotein association. The role of exon 3-encoded sequences, even though highly conserved across species in the case of apoE, is uncertain, since this region is not the primary or major determinant of lipoprotein association and also does not impede lipoprotein association directed by exon 4 sequences.