(Received for publication, August 21, 1995; and in revised form, November 6, 1995)
From the
Cleavage after lysine 32 in the G subtype and
after lysine 36 in the G
subtype of purified mixed
brain G
by endoproteinase Lys-C blocks G
-mediated
stimulation of phosphorylation of rhodopsin in urea-extracted rod outer
segments by recombinant human
-adrenergic receptor kinase
(h
ARK1) holoenzyme while h
ARK1 binding to rod outer segments
is partially affected. This treatment does not attenuate the binding of
the treated G
to C-terminal fragments of h
ARK1
containing the pleckstrin homology domain. Lys-C proteolysis also does
not alter the association of the G
with phospholipids, its
ability to support pertussis toxin-catalyzed
G
/G
ADP-ribosylation, or its ability
to inhibit forskolin-stimulated platelet adenylate cyclase. The G
subunit remains noncovalently associated with the cleaved G
fragments. Thus, in addition to recruiting h
ARK1 to its receptor
substrate, G
contributes secondary and/or tertiary structural
features to activate the kinase.
G-protein-coupled receptor responses to agonist ligands are
modulated at multiple levels along the signal transduction pathway.
Regulation includes short term effects on receptor coupling and on
internalization of the receptor. Longer term effects include
down-regulation of the cellular receptor content and mRNA levels
(Tholanikunnel et al., 1995). Several different protein
kinases have been shown to phosphorylate some of the seven
transmembrane helix receptors on cytosolic portions of the molecule,
reducing coupling of the receptors to their associated heterotrimeric
G-proteins (reviewed by Kobilka(1992)). One family of protein kinases,
the G-protein receptor kinases (GRKs), ()has been defined,
(Premont et al., 1995; Inglese et al., 1993). Three
GRKs have been shown to target agonist-occupied receptors,
phosphorylating multiple serine/threonine residues adjacent to acidic
amino acid residues in the primary amino acid sequence. There are
presently six members of this protein kinase family that share a common
catalytic domain, diverging in the N- and C-terminal extensions outside
of this region.
The GRK2/GRK23 (ARK1 and
ARK2) subfamilies
are encoded by separate genes (Benovic et al., 1991). They
were originally shown to phosphorylate the
-adrenergic
receptor and thus were given the name
-adrenergic receptor kinases
or
ARKs. The
ARKs are C-terminally extended relative to other
members of the GRK family. The C-terminal 222 amino acids of
ARK1
and
ARK2 contain a domain responsible for the association of the
enzyme with heterotrimeric G-protein G
subunits (Pitcher et al., 1992). Addition of G
subunits to an in
vitro phosphorylation system stimulates
ARK phosphorylation
of receptor substrates (Haga and Haga, 1990, 1992), but not that of
peptide substrates (Pitcher et al. 1992). This C-terminal
domain of the kinase includes a region homologous to a domain of the
platelet protein pleckstrin (PH domain) (Touhara et al.,
1993), thought to mediate protein-protein interactions among signaling
proteins (Musacchio et al., 1993; Gibson et al.,
1994; Ingley and Hemmings, 1994). PH domains may be functionally
analogous to the Src homology 2 and 3 domains of tyrosine kinase
signaling systems (Mayer et al., 1993). While the portions of
the
ARK C terminus involved in the association with G
subunits have been delineated (Koch et al., 1993), less is
known about the determinants on the G
partner. The multiple
subtypes of G
and G
, the requirement for the G
heterodimer for cellular function, (Iniguez-Lluhi et al.,
1992), and multiple post-translational modifications (Yamane and Fung,
1993) have impeded study. This paper describes the dissociation of
G
binding to h
ARK1 from G
stimulation of
rhodopsin phosphorylation by this kinase after proteolysis with Lys-C.
G
is cleaved by the protease at lysine 33 in G
(lysine 36 in G
), and the G
fragments
remain associated with the G
subunit.
Frozen bovine retinas were from George A. Hormel, Austin, MN.
Frozen bovine brain was from Pel-Freez, Rogers, AR. ATP, GDP,
GTPS, NAD
, sodium cholate, Lubrol PX,
isobutylmethylxanthine, L-propanolol, phosphoenolpyruvate,
dimyristoyl phosphatidylcholine, forskolin, and pyruvate kinase were
from Sigma. Endoproteinase Lys-C from Lysobacter enzymogenes (catalog no. 476986) was from Boehringer Mannheim. Reagents for
SDS-PAGE were from Research Organics. Nitrocellulose (BA83) was
obtained from Schleicher and Schuell. DE-52 was from Whatman.
Antibodies specific for
,
,
,
, pan-
,
,
,
, and
of the
heterotrimeric G-protein G
and neutralizing peptides were
purchased from Santa Cruz Biotechnology. These antibodies are also
useful for enzyme-linked immunosorbent assay determinations. Antibodies
to the C-terminal 222 amino acids of human
ARK1 were raised
against the glutathione S-transferase (GST) fusion protein
(Cocalico Biologicals, Reamstown, PA) and purified from an IgG fraction
following adsorption against immobilized GST by affinity chromatography
on immobilized GST-C-terminal 222-amino acid human
ARK1, eluting
with 0.1 M glycine, pH 2. Secondary antibody (donkey
anti-rabbit peroxidase) and ECL detection reagents were purchased from
Amersham. [
-
P]ATP (3000 Ci/mmol),
[
-
S]GTP (1045 Ci/mmol), and
[
-
P]NAD
(30 Ci/mmol) were
from DuPont NEN. S-Adenosyl-L-[
H-methyl]methionine
(77 Ci/mmol), the
I-cAMP Scintillation Proximity Assay
Kit, Rainbow prestained molecular weight markers, and Amplify were
acquired from Amersham. Purified recombinant human
ARK His
(PH+C) (Gly
-Ser
) protein
was generously provided by Dr. Daruka Mahadevan (Mahadevan et
al., 1995). DNA encoding the
ARK C-terminal domains
ARK
(PH+C) (Gly
-Ser
) and the
C-terminal 222 amino acids (Pro
-Leu
)
were cloned by PCR from the human
ARK1 cDNA provided by Dr. A.
DeBlasi (Chuang et al., 1992), and their nucleotide sequences
confirmed on an Applied Biosystems model 373A automated sequencer. The
glutathione S-transferase fusion proteins were produced in Escherichia coli strain BL21(DE3) using the pGEX-2T vector
system (Pharmacia Biotech Inc.) and purified according to standard
protocols. Peptides corresponding to residues 8-34 of
G
or residues 3-29 of G
were
kindly provided by Dr. R. Neubig, Department of Pharmacology,
University of Michigan.
Heterotrimeric G-proteins were isolated from
frozen bovine brain, and the G subunit complex (mixed
subtypes of
and
subunits) purified by chromatography in
sodium cholate on heptylamine-Sepharose (Sternweis and Pang, 1990) in
the presence of GDP/AlMgF. Further resolution of the separated
G
from G
subunits for ADP-ribosylation studies was
achieved by ion exchange chromatography on a MonoQ (Pharmacia) column
in 0.1% (w/v) Lubrol PX (Sternweis and Pang, 1990). The purified G
and G
subunits were stored in aliquots at -80 °C.
Two different batches of G
when assayed for effects of Lys-C
proteolysis on rhodopsin phosphorylation and h
ARK1 binding
activity revealed no discernible differences. Carboxymethylation of the
C terminus of the
subunit of G
with S-adenosyl-L-[
H-methyl]methionine
was accomplished using a cholate-extracted brain membrane fraction
(Fung et al., 1990). Bovine retinal rod outer segments (ROS)
containing rhodopsin were purified under red light illumination and
urea-extracted before use as a phosphorylation substrate (Phillips et al., 1989). SDS-PAGE was performed in 10% acrylamide,
0.267% bisacrylamide gels containing 0.1% SDS in the Laemmli buffer
system (Laemmli, 1970). Rhodopsin phosphorylation was determined after
separation of
P-labeled proteins by SDS-PAGE. Radioactive
gels were fixed for 10 min in 25% methanol, 10% acetic acid, washed
with distilled water for 10 min, and the gels dried at 80 °C under
vacuum before exposure to Kodak XAR-5 or X-Omat-LS film. The
P radioactivity of the rhodopsin band was quantitated from
the film using a BioImage (Millipore) scanner. Tritiated proteins
separated by SDS-PAGE were visualized after fixation in 25% methanol,
10% acetic acid, soaking in an Amplify enhancement solution for 45 min,
and drying and exposing to film at -80 °C for 3-7 days.
ARK1 and G
were determined after transfer of polypeptides
separated by SDS-PAGE on a 10% acrylamide, 0.367% bisacrylamide in the
Laemmli buffer system (Laemmli, 1970) to 0.2-µm pore size
nitrocellulose (LeVine and Sahyoun, 1988). An affinity-purified
antibody directed against the C-terminal 222 amino acids of h
ARK1
recognizing the PH domain of that kinase (Sallese et al.,
1995) was used to detect the enzyme. G
subunits were detected with
pan-
subunit-specific antibodies.
and
subunits of the
heterotrimeric G-proteins were separated on 16.5% acrylamide, 0.5%
bisacrylamide gels containing 6 M urea and 0.1% SDS in a
Tris-Tricine buffer system (Schagger and von Jagow, 1987). The subunits
were transferred as described for
ARK1 and were detected with
G
- or G
- subtype subunit-specific antibodies. Quantitation of
the ECL was standardized as described (Mahadevan et al.,
1995).
The G subunits of the heterotrimeric G-proteins
form a heterodimer (
= 35 kDa or 36 kDa,
=
6748-8321 kDa), depending on the subunit subtypes and
post-translational modifications. This non-covalent complex is not
dissociable under non-denaturing conditions and associates reversibly
with an undetermined variety of G
subtypes and with a protein
kinase A substrate, phosducin (Bauer et al., 1992), as well as
a host of effector molecules such as adenylate cyclase, ion channels,
and phospholipases (Clapham and Neer, 1993). The G
s are multiply
post-translationally modified by N termini N-acetylation (Wilcox et
al., 1994) and C-terminal cysteine geranylgeranylation and
carboxymethylation (reviewed by Yamane and Fung(1993)). Although these
modifications are not required for assembly of the G
dimer,
prenylation apparently modulates interactions with other proteins
(Kisselev et al., 1994) and their interaction with the lipid
bilayer (Muntz et al., 1992; Iniguez-Lluhi et al.,
1992). G
requires small amounts of detergent to remain in
solution.
Reducing SDS-PAGE
analysis of the proteolytic product as a function of
protease:G ratio revealed a size range for the G
fragment of M
3400-5200 by silver staining,
a reduction from M
6900-8600, corresponding
to an M
of 3500. There was no discernible effect
on the M
of the G
subunit (Fig. 1).
The mixed G
preparation isolated from frozen bovine brain
contains multiple subtypes of G
and G
subunits, the latter
accounting for the smear around M
6500. Antibodies
raised to synthetic peptides corresponding to the N-terminal 17
residues of the G
subunits or 21 amino acids of the G
subunits (Santa Cruz Biotechnology) are subtype-selective and can be
used to determine the distribution of the subtypes in the preparation.
Western immunoblotting of two separate purifications revealed that the
purified bovine brain G
used in these studies has the
following distribution of G
and G
subunits:
>
>
;
>
(data not shown). This quantitation
assumes that the subtype-selective antibodies are equally capable of
detecting antigen at 1.6 µg/ml antibody. In addition, the
purification of the G
subunits could influence the recovery
and thus the apparent distribution of the different subtypes.
Figure 1:
16% acrylamide, 6 M urea
Tris-Tricine SDS-PAGE analysis of the cleavage of G by Lys-C
(silver-stained). 2 µg of G
were digested with the
indicated ratio of Lys-C protease:G
under the conditions
described under ``Experimental Procedures.'' Lane 1,
no protease; lane 2, 1:400; lane 3, 1:200; lane
4, 1:100; lane 5, 1:50; lane 6, 1:25; lane
7, 1:12.5. The G
and G
subunit positions are indicated
by brackets.
Methylation of the C-terminal cysteine (Cys) carboxyl
moiety of the purified brain G
with S-adenosyl-L-[
H-methyl]methionine
(Fung et al., 1990) provided a marker for the C terminus of
the G
subunit. Lys-C treatment of the tritiated G
released a labeled fragment of M
3500 (Fig. 2), mirroring the shift on SDS-PAGE to lower M
seen with silver staining (Fig. 1). This
implies that the extreme C terminus of the G
subunit remains
intact. Immunoblotting after SDS-PAGE showed that N-terminal G
subunit immunoreactivity (residues 2-17) was eliminated by Lys-C
treatment (Fig. 3). By contrast, for the G
subunit, neither
the N-terminal immunoreactivity (data not shown) nor the size on
SDS-PAGE (Fig. 1) was affected by Lys-C treatment. Gel
permeation chromatography of the
[
H]carboxymethylated G
on a Superose 12
column in 0.8% cholate before or after Lys-C treatment demonstrated
that the fragments of the cleaved G
subunit remained complexed
with the G
subunit (Table 1). When the Superose 12 column
fractions were assayed by enzyme-linked immunosorbent assay and slot
blotting onto nitrocellulose, virtually no loss of the immunoreactive
N-terminal fragment(s) of G
was observed. By contrast,
denaturing SDS-PAGE and subsequent Western blot analysis of the same
fractions revealed a significant loss of N-terminal G
subunit
immunoreactivity (Table 1).
Figure 2:
Reduction in M of
C-terminal [
H]carboxymethylated bovine brain
G
by Lys-C proteolysis visualized by
H
autoradiography. 100 µg of bovine brain G
were
carboxymethylated with 0.5 mg of cholate-extracted bovine brain
membrane protein and 50 µCi of S-adenosyl-L-[
H-methyl]methionine
(77 Ci/mmol) in 0.5 ml of carboxymethylation buffer and the labeled
proteins re-extracted with cholate as described under
``Experimental Procedures.'' Lane 1,
[
H]carboxymethylated proteins incubated for 30
min at 30 °C in the absence of protease; lane 2, + 2
µg of endoprotease Lys-C. The M
24,000 labeled
protein is an endogenous substrate for the carboxymethyltransferase
derived from the brain membrane enzyme source. This contaminant
represents a minor fraction of the
H label incorporated.
Lys-C treatment leads to a M
3500 decrease in
size of the labeled G
subunit. The leading ion front ran to the
very bottom of the gel.
Figure 3:
Removal of N-terminal G subunit
immunoreactivity by Lys-C proteolysis. A total of 10 µg of bovine
brain G
was incubated with the indicated ratio of
endoprotease Lys-C for 30 min at 30 °C. The control lane was
material incubated without Lys-C. The digest was then separated by
Tris-Tricine-urea SDS-PAGE and transferred to nitrocellulose as
described under ``Experimental Procedures.'' The blot was
reacted with a 1:1 mixture of G
-antibodies specific for the
N-terminal
residues(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) of the
G
and G
subtypes and the
immunoreactivity quantitated as referenced under ``Experimental
Procedures.'' The experiment was repeated twice as single
determinations with similar results. A, raw film data. Lane 1, 1:4 (Lys-C:G
(w/w)); lane 2, 1:8; lane 3, 1:16; lane 4, 1:32; lane 5, no Lys-C
added. B, densitometric scan of
film.
Figure 4:
C4
reverse phase chromatography of
[H]carboxymethylated G
subunits.
Fifteen µg of [
H]carboxymethylated
G
were treated with either buffer or 1 µg of Lys-C in a
final volume of 100 µl for 1.5 h at 30 °C. The samples were
dissociated with guanidine hydrochloride and subjected to reverse phase
chromatography as described under ``Experimental
Procedures.'' The
H content of the 0.75-ml fractions
collected was determined by liquid scintillation counting. Circles, incubated without protease; squares, +
1 µg of Lys-C. Arrow, C-terminal G
peptides (see
text); asterisk, undigested G
, G
polypeptides.
Figure 5:
Modulation of ARK1-mediated receptor
phosphorylation. Incubations of G
subunits with ROS and
ARK1 and phosphorylation were performed as described under
``Experimental Procedures.'' A, translocation of
ARK1 to rod outer segment membranes containing rhodopsin. The
amount of
ARK1 binding to light-activated urea-extracted ROS in
the absence, presence of G
subunits, and presence of Lys-C
digested G
subunits was determined after SDS-PAGE by
immunoreactivity with 0.1 µg/ml anti-
ARK1 antibody.
Quantitation of the ECL reaction was by densitometry. Raw film data are
shown at the top. Lane 1, translocation of
ARK1
to ROS in the absence of G
subunits; lanes 2 and 3, Lys-C digested G
subunits; lanes 4 and 5, G
subunits treated with Lys-C in the presence of
10 µg/ml protease inhibitor leupeptin. The experiment was repeated
three times with similar results. B, lack of stimulation of
rhodopsin phosphorylation by proteolyzed G
subunits.
ARK1 phosphorylation of rhodopsin in ROS was determined after
translocation of the kinase in the absence, presence, and presence of
Lys-C-digested G
subunits. Following SDS-PAGE, the
P incorporated into rhodopsin was quantitated by
densitometry of the autoradiogram. C, dose response of
G
subunits on rhodopsin phosphorylation. ROS were
phosphorylated with
ARK1 and the indicated concentrations of
control incubated or Lys-C digested G
subunits as indicated
under ``Experimental Procedures'' without removing unbound
proteins. Following SDS-PAGE the
P incorporated into
rhodopsin was quantitated by densitometry of the autoradiogram. Circle, control incubated G
subunits; square, Lys-C digested G
subunits. Data are single
determinations, and the experiment was repeated twice with similar
results. The raw autoradiographic data are shown at the top.
The first five lanes are G
subunits treated with
Lys-C in the presence of 10 µg/ml leupeptin, while the final
four lanes are Lys-C-digested G
subunits. Lane
1, no G
subunits; lanes 2 and 6, 27.9
nM G
subunits; lanes 3 and 7, 55.8
nM; lanes 4 and 8, 112 nM; lanes 5 and 9, 223
nM.
Figure 6:
Effect of Lys-C proteolysis of G
subunits on binding to the isolated PH domain of
ARK1. The
indicated concentrations of treated G
subunits were incubated
in a 50-µl reaction volume with 0.5 µM GST-
ARK1
C-terminal (Pro
-Leu
) fusion protein
immobilized on glutathione-Sepharose as indicated under
``Experimental Procedures.'' The beads were washed and
subjected to SDS-PAGE, and the proteins transferred to nitrocellulose
for immunodetection of the G
with a pan-G
-specific antibody. A, raw film data. Lanes 1, 5, and 9, 106 nM G
subunits; lanes 2, 6, and 10, 319 nM; lanes 3, 7, and 11, 957 nM; lanes 4, 8, and 12, 2874 nM. Lanes 1-4 represent G
subunits incubated on ice, lanes
5-8 are G
subunits treated with Lys-C at 30 °C
for 30 min in the presence of 10 µg/ml leupeptin and lanes
9-12 are G
subunits treated at 30 °C with
Lys-C for 30 min. B, quantitation by densitometry. Circle, G
subunits incubated on ice; square, G
subunits treated with Lys-C at 30 °C
for 30 min in the presence of 10 µg/ml leupeptin to inhibit the
protease; triangle, G
subunits treated at 30 °C
with Lys-C for 30 min. Data are representative of four experiments with
single determinations.
Figure 7:
Functional assay of truncated G
subunits. A, G
subunit-mediated ADP-ribosylation of
G
/G
by pertussis toxin. The reaction
was carried out as described under ``Experimental
Procedures.'' Results are expressed as average of duplicates
± S.D. The experiment was repeated twice with similar results. Circle, G
subunits incubated on ice; square, G
subunits treated with Lys-C at 30 °C
for 30 min in the presence of 10 µg/ml leupeptin to inhibit the
protease; triangle, G
subunits treated at 30 °C
with Lys-C for 30 min. B, inhibition of forskolin-stimulated
platelet type I adenylate cyclase by G
subunits. The effect
of treated G
subunits on platelet membrane adenylate cyclase
activity was determined as described under ``Experimental
Procedures.'' Circle, G
subunits incubated on
ice; square, G
subunits treated with Lys-C at 30
°C for 30 min in the presence of 10 µg/ml leupeptin to inhibit
the protease; triangle, G
subunits treated at 65
°C for 10 min to inactivate the proteins. Data shown are the
average of triplicates ± S.D. Basal adenylate cyclase activity
= 200 pmol/min/mg protein, forskolin-stimulated adenylate
cyclase activity (100%) = 2200 pmol/min/mg protein. The
experiment was repeated twice.
G subunits participate in a diverse range of
biological interactions, from anchoring and modulating the GTPase
activity of G
subunits, to activation or inhibition of
transmembrane signaling effectors such as phospholipase C
and
isozymes, phospholipase A
, adenylate cyclase
isozymes, and ion channels (reviewed by Clapham and Neer(1993)). They
are also critically involved in regulating the activity of several of
the G-protein coupled receptor kinases (GRK2 and GRK3) toward receptor
substrates (Inglese et al., 1993). The multiple isotypes
(5-
and 7-
isotypes) of G
subunits provide a
daunting array of possible combinations, which may be significant
(Kleuss et al., 1993; Kleuss et al., 1992). The
assembly of the dimeric structure is further complicated by their
subsequent extensive post-translational modification and proteolytic
processing. Chimeric studies in COS7 cells taking advantage of the
differential interaction of G
with G
but not G
, while G
will
interact with both G
s (Simonds et al., 1991), defined
multiple sites of interaction of the
subunit with the
subunit, and assigned subtype selectivity to G
residues 215-340 (Garritsen and Simonds, 1994) or residues
210-293 (Katz and Simon, 1995). A series of G
truncation experiments suggest that a region between residues
45-59 in the G
subunit are involved with dimerization with
G
(Mende et al., 1995).
At high
concentrations trypsin will cleave the G subunit at
Arg
, generating two proteolytic fragments of M
26,000 and 15,000 without noticeable effect on
the G
subunit in native G
subunits (Tamir et
al., 1991). The accessibility of the cleavage site in the
subunit to Lys-C proteolysis demonstrated in the present work
illuminates the quaternary structure of the G
dimer. To
minimize possible interactions of the prenyl moiety with lipid bilayers
and potential steric hindrance, the proteolysis was performed in
cholate, an anionic detergent with the small aggregation number of 2
cholate molecules/micelle (Calbiochem, 1993). There are 7
(G
) or 6 (G
) lysines in the G
subunit, which could potentially be available for Lys-C proteolysis.
The major identified G
products of this protease begin with
Ala
(G
) and Ala
(G
). Monitoring of the migration of the
C-terminal [
H]carboxymethylated G
on HPLC
gave no evidence for specific smaller or larger cleavage products.
Thus, the majority of the treated G
retains the prenylated
C-terminal cysteine residue. This part of the molecule is thought to
interact with the seven-transmembrane helix receptor in the fashion of
transducin of the visual system. The farnesylated C terminus of
G
, stabilizes the active
MII form of rhodopsin, which activates G
(transducin) in
the presence of GTP (Kisselev et al., 1994). Hints as to the
relative positioning of the
and
subunits come from several
sources. Copper o-phenanthroline-mediated cross-linking of
G
and G
through proximal
intersubunit cysteine residues in transducin indicates that
Cys
and
Cys
or Cys
are
nearby in the three-dimensional protein structure (Bubus and Khorana,
1990). This was interpreted as a point of close contact between the
subunits. Alignment of transducin G
with G
and G
places the reactive
Cys
/Cys
at the site of Lys-C digestion
determined here. Perhaps lysines C-terminal to this position are not
accessible to the protease because of
subunit-
subunit
interactions or interactions involving the prenyl group. A series of
sequential amino acid replacements in G
(35-37)
and G
(38-40) are sufficient to specify the
appropriate
-
selectivity (Lee et al., 1995).
Interestingly, this motif is immediately C-terminal to the Lys-C
cleavage site.
G subunits have been shown to mediate GRK2
and GRK3 (
ARK1 and
ARK2) translocation to retinal ROS
containing the light receptor substrate rhodopsin, cell membranes
(Pitcher et al. 1992), or to phospholipid vesicles (Kim et
al., 1993). In addition, they robustly stimulate the activity of
the kinase 10-20-fold toward receptor substrates but much less so
toward synthetic peptide substrates (Pitcher et al. 1992).
Cleavage of the G
-
complex with Lys-C retained the two
fragments 1-32(36) and 33(37)-68(71) non-covalently associated
with the G
subunit. Tryptic fragments of the G
complex
cleaved within G
similarly remain attached (Fung and Nash, 1983;
Thomas et al., 1993). G
is not cleaved by trypsin in the
G
complex (Tamir et al., 1991). The Lys-C
G
fragment complex retained the ability to function as a
binding site for the PH domain of
ARK1, to productively associate
with G
/G
to allow those proteins to
serve as pertussis toxin substrates, and to inhibit Type I adenylate
cyclase activity. On the other hand, the cleaved complex only weakly
stimulated
ARK1 phosphorylation of rhodopsin. Lys-C cleavage may
not be unique in diminishing G
stimulation of rhodopsin
phosphorylation by
ARK1. The effect of trypsin cleavage of G
has not been assayed.
The inability of N-terminal G or G
peptides or immunoprecipitating antibodies to these peptides to affect
G
-dependent rhodopsin phosphorylation or binding to the PH
domain suggests that a region of the G
other than the N
terminus is involved in these interactions. This suggestion is
consistent with the findings of Wang et al.(1994) that
ARK PH domain binding may be mediated through the C-terminal five
WD40 motifs of G
rather than through G
.
There may be
multiple points of interaction between G,
ARK, and other
molecules. Some regions may specify binding to target molecules while
others may modulate the catalytic activity of
ARK in conjunction
with the ligand-activated receptor substrate. The modulation of
ARK activity could also be occurring at the level of G
interaction with the receptor substrate. While the fragments of
Lys-C-digested G
appear to remain non-covalently associated in
solution as the G
dimer, either they are displaced in the
receptor complex with G
, or they fail to assume the
appropriate relationship to the rest of the members of the complex to
activate
ARK phosphorylation of the receptor. Nicking of the
G
subunit by proteolysis with Lys-C indicates that the G
dimer provides active modulation rather than a passive kinase binding
scaffold for the phosphorylation of G-protein-coupled receptor
substrates by
ARK1.