(Received for publication, March 3, 1995; and in revised form, June 7, 1995)
From the
With the growing awareness that the G protein and
subunits directly regulate the activities of various enzymes and ion
channels, the importance of identifying and characterizing these
subunits is underscored. In this paper, we report the isolation of cDNA
clones encoding eight different human
subunits, including three
novel forms designated
,
, and
. The predicted protein sequence of
shares the most identity (60-77%) with
,
, and
and the least identity (38%)
with
. The
is modified by a
geranylgeranyl group and is capable of interacting with both
and
but not with
.
The predicted protein sequence of
shows only modest
to low identity (35-53%) with the other known
subunits,
with most of the differences concentrated in the N-terminal region,
suggesting
may interact with a unique subclass of
. The
is modified by a geranylgeranyl group and
is capable of interacting with
and
but not with
. Finally, the predicted protein
sequence of
shows the most identity to
(76% identity) and the least identity to the other known
(33-44%). Unlike most of the other known
subunits,
is modified by a farnesyl group and is not capable
of interacting with
. The close resemblance of
to
raises intriguing questions
regarding its function since the mRNA for
is
abundantly expressed in all tissues tested except for brain, whereas
the mRNA for
is expressed only in the retina where
the protein functions in phototransduction.
Intracellular transmission of extracellular signals are most
commonly mediated by a family of guanine nucleotide-binding proteins (G
proteins) that couple with various receptors and effectors to produce
appropriate cellular responses. The G proteins are heterotrimers,
composed of ,
, and
subunits. In response to binding of
the appropriate ligand, the receptor stimulates the exchange of bound
GDP for GTP on the
subunit, resulting in the dissociation of the
subunit from the
and
subunits. The GTP-bound
subunit has been shown to directly regulate the activity of downstream
effectors (1, 2, 3) . Recently, it has been
shown that the
subunits, which exist as a tightly associated
complex in vivo(1) , can also regulate the activity of
a specific subset of downstream effectors, including adenylyl cyclase
subtypes II and IV, phospholipase A2, phospholipase C subtypes
1,
2, and 3, and K
and Ca
channels(4, 5, 6) . Thus, the G protein
and
subunits produce bifurcating signals that regulate
effector function. Moreover, the
subunits can directly bind
to receptors (7) and can increase agonist-dependent
phosphorylation and desensitization of receptors by directly
interacting and recruiting the
-adrenergic receptor kinases to the
membrane(8, 9) . Thus, the
subunits play
prominent roles in both effector regulation and receptor recognition.
As the number of
,
, and
subunits continues to grow,
the task of unraveling the subunit composition and function of
individual G proteins is becoming more complex.
Both the and
subunits belong to large multigene families. Complete cDNAs
encoding five distinct mammalian
subunits
(
-
) have been identified thus
far (10) . A rat heart cDNA identified recently may encode a
sixth
subunit, which is 96% identical to the human
subunit (11) . At the amino acid level, the
subunits are highly conserved. In contrast, the
subunits are much
more divergent, suggesting this may determine the functional
specificity of the
subunit complex. Complete cDNAs
representing five different
subunits have been reported with the
isolation of the
subunit from bovine
retina(12) , the
,
, and
subunits from bovine
brain(13, 14, 15, 16) , and the
subunit from bovine and rat liver (17) . (
)The existence of a putative
subunit has
also been reported with the isolation of a PCR (
)fragment
from mouse kidney and retina (15) . In the present paper, we
report the isolation and characterization of cDNA clones encoding the
human homologs of the five known
,
,
,
, and
subunits as
well as three previously unknown
,
,
and
subunits. (
)Comparison of the
,
, and
subunits
reveals some interesting amino acid homologies. Of particular interest,
the
subunit shows only a low level of homology
(35-53%) with the other
subunits, suggesting the existence
of a new subclass of
subunits. On the other hand, the
subunit shows a high level of homology (76%) to the
subunit. This close resemblance to the
subunit raises important questions regarding the function of the
subunit since the mRNA for
is
expressed in a wide variety of tissues, whereas the mRNA for
is expressed only in the retina where it functions in
phototransduction. In addition to presenting their cDNA and deduced
amino acid sequences, we examine the tissue distribution of the
,
, and
subunits
and show their selective interactions with the
,
, and
subunits.
The complete nucleotide and deduced
amino acid sequences of the newly identified ,
, and
subunits are shown in Fig. 1Fig. 2Fig. 3. In the case of the
subunit, partial cDNA clones were isolated from various cDNA
libraries, including jurkat T-cell, bone marrow, prostate, 6-week-old
embryo, and adrenal gland tumor libraries, by EST sequencing. The
longest 689-bp cDNA reported here was isolated from the adrenal gland
tumor library. The cDNA includes 98 and 365 bp of 5`- and
3`-untranslated (UTR) sequences, respectively (Fig. 1). The
first ATG codon at position 99 has the characteristics of a translation
initiator codon with the expected purines at positions -3 and
+4(23) . A second ATG codon at position 111 lacks the
expected purines, making it less likely to be the initiator codon. A
typical polyadenylation signal (AATAAA) was not found, but a
polyadenine sequence was observed near the 3`-end of the cDNA. In the
case of the
subunit, several partial ESTs were
identified in a variety of human cDNA libraries, including T-cell
lymphoma, fetal heart, stimulated monocyte, osteoclastoma, stromal cell
line TF274, placenta, neutrophil, bone marrow, prostate, and
hippocampus. The longest 1213-bp cDNA was isolated from the T-cell
lymphoma library. The cDNA includes 23 and 986 bp of 5`- and 3`-UTR
sequences, respectively (Fig. 2). The long 3`-UTR possesses a
poly(A) tail, a polyadenylation signal toward the 3`-end, and several
A(T)
A motifs implicated in mRNA stability(24) . In
the case of the
subunit, partial cDNA clones were
detected in a number of human libraries, including pineal gland,
testes, thymus tumor, stromal cell line TF274, 6-week-old embryo, and
platelet-derived growth factor-induced endothelial cell libraries. The
longest 654-bp cDNA reported here was isolated from the testes library,
which includes 106 and 326 bp of 5`- and 3`-UTR sequences, respectively (Fig. 3). The 3`-UTR contains a polyadenylation signal and a
poly(A) tail toward the 3`-end.
Figure 1:
Nucleotide and predicted amino acid
sequence of the human subunit. The first ATG codon of
the open reading frame starts at position 99. Amino acids are denoted
by single-letter codes.
Figure 2:
Nucleotide and predicted amino acid
sequence of the human subunit. The open reading
frame starts at position 24. Amino acids are denoted by single-letter
codes. A potential polyadenylation signal (AATAAA) is underscored with solid lines.
Figure 3:
Nucleotide and predicted amino acid
sequence of the human subunit. The open reading
frame starts at position 108. Amino acids are denoted by single-letter
codes. A potential polyadenylation signal (AATAAA) is underscored with solid lines.
Figure 4:
Comparison of the human subunits. An
alignment of the human
,
,
,
,
,
,
, and
was made
with the GCG PILEUP program. Regions I, II,
and III are overlined and represent the
-
interaction region,
-
interaction region, and CAAX box, respectively (see text for explanation). Regions A, B, and C represent regions that are highly conserved
in all
subunits. Identical amino acid residues are boxed. Amino acid differences between the human and bovine
and
subunits are underlined.
Most of the
homology among the subunits was concentrated in several discrete
regions (Fig. 4). The N-terminal region of the
,
, and
subunits
is the most divergent at the amino acid level (region I), consistent
with the newly defined role of this region in determining the
specificity of the interaction between the
and
subunits(25) . An internal region of 14 amino acids, which has
been implicated in determining the specificity of the interaction
between the
and
subunits(26) , is conserved to
varying degrees between the
,
, and
subunits (region II). Finally, the C-terminal region
containing the CAAX sequence (C = cysteine; A =
aliphatic; X = leucine, serine, or methionine), which
has been shown to direct prenylation and carboxyl-methylation of these
proteins(27, 28) , is conserved in the
,
, and
subunits
(region III). Three other regions that are highly conserved in
mammalian and Drosophila
subunits (29) are also
conserved in the
,
, and
subunits (regions A, B, C). Although their roles
have not yet been established, these regions may be important in
generating similar conformations of the
subunits.
Figure 5:
Prenylation of the ,
, and
subunits in vitro.
Translations were performed in the TNT-coupled rabbit reticulocyte
lysate system in the presence of [
S]methionine (panelA) and [
H]FPP or
[
H]GGPP (panelB).
Figure 6:
Northern blotting analysis of the
,
, and
subunits.
Each lane represents 2.0 µg of
poly(A)
-enriched mRNA from different human tissues
(Clontech). Lane 1, heart; lane 2, brain; lane
3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane
8, pancreas. The positions of RNA size markers are indicated on
the right. The blot was hybridized sequentially with
radiolabeled probes specific for
(panel A),
(panel B), or
(panel
C), respectively. Blots were exposed at -80 ° for 4 days
for
and overnight for
and
.
As shown in Fig. 7, the in vitro translated ,
, and
monomers were almost
completely digested by trypsin (panelsA-C). In
contrast, in vitro translated mixtures of the
subunit and either the
,
,
, or
subunit yielded a 26-kDa
protected fragment when digested by trypsin under identical conditions (panelA). Likewise, in vitro translated
mixtures of the
subunit and either the
,
, or
subunit
produced a 26-kDa protected fragment when digested by trypsin (panelB). However, when an in vitro translated mixture of the
subunit and the
subunit was digested by trypsin, no such fragment
was generated (panelB), even though the levels of
the
subunits and the
subunits in the in vitro translated mixtures were comparable. Taken together, these results
indicate that the
subunit is able to form dimers with
the
,
, and
subunits, whereas the
subunit is able to form
dimers with the
and
subunits but
not with the
subunit. The inability of the
subunit to form a dimer with the
subunit is particularly interesting since the
subunit most closely resembles the
subunit at
the amino acid level, and the
subunit is unable to
form a dimer with the
subunit(30) . Finally,
no protected fragment was generated when in vitro translated
mixtures of the
subunit and the
,
,
, or
subunit
were digested by trypsin (panelC). However, this
result is more difficult to interpret since no positive control exists
for the
subunit at the present time. Thus, it is not
certain whether this result indicates that the
subunit is not able to form dimers with any of the known
subunits or whether the
subunit is able to form
dimers with some of the known
subunits but that tryptic digestion
of the resulting
dimers does not generate a
protected fragment of
. In this regard, the
subunit has been reported to contain one more
potential tryptic digestion site (lysine 177) in the 26-kDa fragment
than the
or
subunits(30) .
Thus, if trypsin cleaves at lysine 177 in addition to arginine 129,
then a 26-kDa fragment of the
subunit may not be
observed. To rule out this possibility, in vitro translated
mixtures of the
subunit and the
,
,
, or
subunits
were digested by Arg-C, a protease that cleaves only at arginine
residues (data not shown). However, again, no protected fragments of
the
subunit were observed, consistent with the
interpretation that the
subunit is not able to form
dimers with any of the known
subunits. Taken together, these
results indicate that the
,
, and
subunits have the ability to selectively associate
with particular
subunits, consistent with previous results on the
,
,
,
, and
subunits(30, 31, 32, 33) . In
this regard, an examination of the amino acids that are common to the
and
subunits, but are not common
to the
,
, and
subunits, may shed further light on the regions of the
subunit that are important for forming dimers with the
subunit.
Figure 7:
Tryptic analysis of -
dimer
formation. In vitro translations were performed in the
presence of [
S]methionine. Subsequently, in
vitro translated
monomers or cotranslated
-
mixtures were incubated in the absence or presence of trypsin, as
indicated. The arrowhead indicates the position of the 26-kDa
protected fragment of
.
Among
members of the subunit family, there are marked differences in
the tissue distribution. Some members, such as the
,
,
, and
subunits,
are restricted to one or a few tissues, whereas others, such as the
,
,
, and
subunits, are expressed in a wide variety of tissues (16) . Furthermore, in most cell types within a tissue, only a
certain subset of
subunits is present(35, 36) .
It is likely that such differences in distribution may be important in
limiting the number of combinatorial associations of the
,
,
and
subunits into functionally distinct G proteins. In this
regard, differences in the subcellular localization of various
subunits have also been reported(37) . It is likely that
particular
and
subunits will be found to share these
patterns of subcellular localization in future studies.
The functional
significance of adding a geranylgeranyl versus a farnesyl
group has not been addressed for this family of proteins. However, the
idea that different types of prenyl groups impart distinct functional
properties is suggested by analysis of retinal and brain
subunits. In particular, striking differences between the retinal and
brain
subunits have been reported in terms of membrane
association(39) , interaction with G protein
subunits(25) , receptors(40) , receptor
kinases(9) , and effectors(32) . Since the retinal and
brain
subunits share a common
subunit,
these differences would appear to be due to their unique
subunits. In this regard, the retinal
subunits contain a
farnesylated
subunit, whereas the brain
subunits are composed mainly of a mixture of the geranylgeranylated
,
, and
subunits.
Consistent with the idea that farnesyl groups are less hydrophobic than
geranylgeranyl groups, the retinal
subunits can
be readily eluted from membranes at low ionic strength(39) ,
whereas the brain
,
, and
subunits require detergents to be eluted from
membranes. However, it is not yet known whether the difference in
membrane association is due to a difference in the primary structures
of the
subunits, the nature of the prenyl group added to the
proteins, or some combination of both. In this regard, it will be
interesting to examine the
subunit. Since the
subunit shares several unique structural features of
the
subunit that are not observed in any other
subunit, including modification by a farnesyl group, we predict that
the
and
subunits may represent a
unique subclass of the
subunit family that interacts reversibly
with the membrane.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31382[GenBank], U31383[GenBank], and U31384[GenBank].