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protein-coupled receptors (GPCRs) signal through multiple pathways that are regulated by G proteins and
-arrestins.1,2 Many of these signaling pathways respond selectively to ligands that are able to stabilize preferred subsets of
receptor conformations.3,4 As a result of conformational heterogeneity, small differences in ligand structure can dramatically shift
signaling toward one response pathway and away from another.5
The ability of an agonistreceptor pair to produce a quantitative
response, measured as efficacy, has been historically modeled
by a transducer ratio parameter reflecting the total receptor
concentration and the transduction of the agonistreceptor
complex into a pharmacological response.6 Potent ligands
having low transducer ratios may not be efficacious, and conversely, efficacious responses precipitated by large transducer
ratios do not necessarily require potent ligands. Because ligand
potencies and their associated transducer ratios can vary widely,
a signaling bias may result in which different ligands produce
variable degrees of response in a single pathway or a single
ligand displays large differences in efficacy between two independent signaling pathways.7
A comprehensive review of qualitative and quantitative
strategies for assessing ligand bias is found in ref 8. The available approaches similarly address bias within the confines of
experiment and attempt to define it observationally or numerically, by data trends or bias factors, as a property that arises
from the signaling paradigm. In contrast, an axiomatic formalism for bias could be developed in a manner that is independent of experiment and subsequently applied to a particular
signaling paradigm. We believe that this latter approach allows
2012 American Chemical Society
Biochemistry
Article
conjugate to
a
f
1 B12
1
=
=
.
f+g
g /f + 1
2
Functions f and g Are Chosen as Rectangular Hyperbolae9
with Different Hill Coefficients. For unequal Hill coefficients
(j j), set
j
Then
k j
j
P
c j
z = x j = , = , = , and = 1
k
k
j
P2
a
Then the bias B12
, which equals
(1 f / g )
becomes
(1 + f / g )
1 z(1 )
a
B12
(1 )
1 + z (z
+ z)
1 + z + (z(1 ) + z)
(1 )z
1 + z(1 )
1 + z(1 )
(1 + )z
(1 + z(1))
+1
a
B12
in Terms of Conjugate Variables B0 and B. Important
a
conjugate relationships we will employ to express bias B12
involving parameters and are:
B =
(1 )
(1 )
and B0 =
.
(1 + )
(1 + )
In terms of B0,
1 z
B0 +
(1)
1 + z(1)
1 z(1)
1 + z(1)
1 + B0
1 z(1)
1 + z(1)
(1 + )z
1 + z(1)
1 z(1 )
B0 +
1
y
1 + z(1 )
a
B12
=
,
B
,
1 + B 1 z(1 )
1 + y 1 + y
0
1 + z(1 )
a
B12 in Terms of B0 and B for Equal Hill Coefficients. In
this case, = 1 and the bias has the simple form:
1
B0
B y
y
a
,
B12
= (B0 , B)
+ ,
=
1+y
1 + y 1 + y 1 + y
THEORETICAL CALCULATIONS
a
Form of the Bias Function B12
. Conjugate Relationships. For bounded, continuous functions f and g over the
interval (c1, c2), f(c), g(c) 0, the bias function is defined as
where y =
gf
1a
. Note that for relationships of the form b =
,
1+a
g+f
(1 + )z
and z =
(1 + )
c j
.
k
()
a=
a
g
1 + B12
1
=
=
, and
f+g
f /g + 1
2
a
f
1 B12
=
a ,
g
1 + B12
a
B12
=
f
and
g
1f/g
1b
a
. Therefore, when
is defined, then B12
is
1+b
1+f/g
P=
1115
E()
K
=
and for EC50, k = A where KA is the
+1
Em
+1
dx.doi.org/10.1021/bi201308s | Biochemistry 2012, 51, 11141125
Biochemistry
Article
1/(1 + 1)
K /( 2 + 1)
K
; = A2
, = A2 .
2/( 2 + 1)
KA1/(1 + 1)
KA1
y changes from 0 to ,
y
1
+
= 1. A plausible relationship is one in
1+y
1+y
y
1
= cos2x ,
= sin 2x , because their sum is 1;
which
1+y
1+y
b
Form of the Bias Function B12
(equal Hill coefficients).
From the derivations given above for equal Hill coefficients:
1, and
a
a
a 2
a 2
g
f
1 + B12
1 B12
(1 + B12
) (1 B12
)
=
a
a =
a
a
f
g
1 B12
1 + B12
(1 B12 )(1 + B12 )
(1
a
4B12
a
B12 )(1
a
+ B12
)
and
g
f
+z
1
1+z
.
=
f
g
+z
1
1+z
y = tan1 y ,
Y y =
2 2
|| A ||
B
tan (B) = = constant .
B0
M( , B , y ) = < BY >
1
y
BY = (B0 + iB)
i
1+
1 + y
B
B
B y
= 0 + + i
1+y
1+y
1 + y
The term
B0 y
.
1 + y
cos(B)
cos(B)
= 1.
cos(B)
= 1 of M(,B,Y) preserves the
cos(B)
cos(B) iB
e
cos(B)
1
(cos y , sin y)
cos y + sin y
x 2 + y2 .
, ||(x , y)|| =
|| C ||
B = B0 + iB and Y =
= sin y ,
1
5
7
3
, B
and
B
4
4
sin
4
4
|
B
R max
=
B
1 , otherwise
|cos |
C
= || A || cosx C and
between them. Thus, <A C >
1 + y2
B0
(1 x y )
A
<A C >
= cos y ,
1 + y2
B
tan B = tan , each co-ordinate point (B0,B) along the B
1 + B 1/ j
=
EC50 .
1 + B0
2(dx 2 + dy 2 )
[1 + ] 1/ j
1 + c j
1=
and, C1/2 =
EC50
1 + k
(1 + )
ds 2 =
y
1
goes from 1 to 0,
from 0 to
1+y
1+y
B0
B y
+
1+y
1+y
a
Because the bias function B12
is always between 1 and 1, the
inner product of B and Y in vector or complex number notation
Biochemistry
Article
, and the length of that vector is the bias and the vector
d(0, R ) = ln
Distances and Mapping in the Unit Disk and BiasCylinder. Hyperbolic Distance between Pairs of Complex
Points, z1 and z2, in the Poincare Disk. This distance is:
z z2
d(z1, z 2) = 2tanh1 1
= ln
1 z1 z 2
1+
1
z1 z 2
1 z1 z 2
2m 1
this distance is:
2m + 1
2m
d0, m
2 +
10, 11.
z1 z 2
1 z1 z 2
d(z1, z 2) = ln
1
d(z1, z 2) = ln
a1 a2
1 a1a2
a1 a2
1 a1a2
1+
; ln
1
b1 b2
1 b1b2
b1 b2
1 b1b2
g
f
, or
1 zz1
(1 + h )(1 h )
1
2
d(z1, z 2) = ln
(1 h1)(1 + h2)
1 (1 )
(a , b) = (B0 , B) =
,
and,
1 + (1 + )
c1 =
1
1
2
d(B01, B02 ) = ln 1 1 and d(B
, B
) = ln .
2 2
2
Let z be the set of all points in the disk about z1 such that d(z,z1)
g
g
dz1. From above, ed(0,z1) = 1 and ed(0,z 2) = 2 . Therefore,
f2
f1
g2 d(z , z ) g2
g1 d(z , z )
e 1 2,
e 1 2,
f1
f2
f2
f1
g1 d(z , z )
g2
g1 d(z , z )
1 2
e
e 1 2 , and
f1
f2
f1
g1 d
g2
g1 d
g1
1
0.69,
log 2e
m
m
B0m = (B0m, 0) and B
= (0, B
), m = 1, 2:
w=
gf
2m 1
= m
and
g+f
2 +1
g
2m
=
f
1
1
= cm.
1
a
Observe that in model B12
we can set R =
1+R
1
1+R
.
=
log 2
1R
log 2e
1R
(dy)2
cosy 4
f1
z1
f2
f1
e z1;
1117
f1
B0
Biochemistry
Article
z1 = bias(B0 , B, 1, 2)
bias(B0 , B, 1, 2) =
and
cos(B) iB
e ,
cos(B)
so that,
where
1
2
, B
dM B01, B , , M B02 , B , = d(B
)
2
2
= ln 1
2
bias(B0 , B, )w()d
w()d = 1.
Bias Factor Expression in Heuristic Modeling.8 Equiactive Comparison. From Bias Viewed as a Parametric
Mapping from a Square to a Disk and Distances and Mapping
in the Unit Disk and Bias Cylinder,
M( , B , 0) = < (B0 , B)(1, 0) >
=
/ ( + 1)
and = i i
. Identifying i and j with pathways and 1
ilig jlig
(ilig + 1) (jlig + 1)
lig
1
2
d(B
, B
) = ln =
ref .
ref
j
ref i
(iref + 1) ref + 1
(
)
cos(B) iB
e
cos(B)
cos(B)
B0eiB
cos(B)
1
2
Setting = d(B
, B
) we see that for small transducers for w hich < 1 this result reduces to
lig
path1
path2
= l ig
l ig , lig = ln ref , which is the result
found in ref 8 (within a multiplicative constant). The bias
factor defined in ref 8 is appropriate for small transducers.
However, for very large transducers in both pathways, this bias
factor should in general approach zero, and this is not
lig
guaranteed to occur with defined using lig = ln ref . If
the reference ligand is unbiased, then ref = 1 and the bias factor
1
is d(B
,0) = ln(lig).
Curve Fitting of Bias Parameters for Bias-Coordinate
Distance Determinations. Using the definitions of the
M(B01, B , 0) M(B02 , B , 0)
=
cos(B) 1 iB
cos(B) 2 iB
B0 e
B0 e
= B01 B02 ,
cos(B)
cos(B)
so that,
d(M(B20 , B , 0), M(B20 , B , 0)) = d(B01, B02 ) = ln
with =
Pikj
k iPj
11
2 2
coordinate B01, and ref with coordinate B02, we obtain the bias
factor:
= d(B01, B02 )
Emax,process i EC50,process j
= ln
EC
E
50,process i max, process j lig
1 1 + iw
ln
and hyperbolic arctangent
2i 1 iw
1
1 1 + w 12
Arth(w) = Atctg(iw) = ln
, the above mapping is
i
2 1w
arctangent as Atctg(w) =
= 2 Arth(bias) =
cos(B)
BeiB.
cos(B)
Y = Atctg(y) =
1
2
M B
, B , M B
, B ,
2
2
=
2
1 + bias
Atctg(i bias) = ln
i
1 bias
1+i
B
B0
1
ln
, B0 0
=
Arctg
B
2i
B0
1i
B0
B =
B0 < 0
Arctg B + ,
cos(B) iB
2
cos(B)
=
j / j + 1
1 + iy
1
ln
2i
1 iy
y=
1 + B0 c j
1 + B k
for ( = 1)
The bias co-ordinates in rotated or non-rotated (n = 1 or 0,
respectively) complex notation and Euclidean notation are:
(x, iy) = bias ei(BnY), and (x, y) = bias[cos(B nY), sin(B
n Y)]. Determining the bias parameters from experiment
requires fitting to concentration c and calculating Y, which
cos(B) 1 iB
cos(B) 2 iB
Be
Be
cos(B)
cos(B)
2
,
= B1 B
1118
Biochemistry
Article
1
,
1 B0 (1 )
1 +
Yc
1 + B0
(1
)
1 Yc
1
B0 +
(1 )
Yz
1
Yc
+
bias =
, B0 tan B
,
(1 )
1 + Yz 1 + Yz
1 + B 1 Yc
0
1 + Yc(1 )
1 + B0 tan B
2
1
,
R1(c1) + R2(c2)
1
1
+
,
R1(c1)
R2(c2)
Fitting Macro:
R1(c1) + R2(c2)
Yc = 10 j(XK)
Y = Yc(1 )
Yz = 2Yc/([1 + B0 tan(B)]{1 + [(1 B0)/(1 + B0)]Y})
Y1 = 1/(1 + Yz)
Y2 = Yz/(1 + Yz)
B1 = {B0 + [(1 Y)/(1 + Y)]}/{1 + B0[(1 Y)/
(1 + Y)]}
B2 = B0 tan(B)
Y = B1Y1 + B2Y2
b (R , R ) = R2(c2) R1(c1)
(b) B12
.
1 2
R1(c1)
R2(c2)
RESULTS
Overview of Strategy. An analysis of biological signaling
bias will be presented in four parts. First, a general bias
formalism will be developed with paradigms showing
procedures that input response functions and output biasresponse functions. Second, the number of candidate paradigms
will be narrowed to two. Third, a particular class of biological
response functions will be chosen and processed by each
of the bias paradigms for generation of the corresponding
bias function. Last, the bias-response functions will be
characterized and applied to the analysis of dopamine
receptor 2 signaling. Mathematical detail that is not necessary
for moving the main discussion forward has been placed in
Theoretical Calculations.
Development of Bias Formalism. We now define the
properties of a bias response function using a generating function B whose domain includes response functions that are
bounded, positive, and continuous. B acts upon paired response
functions and by a series of rules creates a corresponding bias
function. Specifically, the function B(R1,R2) compares the
signaling of response functions R1 and R2 that represent two
processes, A1 and A2, with respective inducers c1 and c2. R1 and
R2 are each standardized by normalization using a maximally
efficacious inducer, c1m and c2m, producing responses E1m and
E2m, respectively. Thus, the relative response [En(cn)]/Em of all
other inducers (n m) of either R1 or R2 is 1.
Because bias suggests preference, a function B(R1,R2) for an
ordered pair of responses should quantitatively predict an
opposite bias exists for the reverse ordered pair, i.e., B(R1,R2) =
B(R2,R1). Functions with this property are defined as odd as
opposed to even functions G with the behavior G(R1,R2) =
G(R2,R1). Additionally, bias implies some difference exists
between two responses R1 and R2, and a simple relationship to
reflect difference is subtraction. This leads to a generating
E (c )
P
=
kj
Em
1+ j
c
(I)
Biochemistry
Article
a
Figure 1. Theoretical bias curves. (A, C, and E) Curves for model B12
were generated from parameters B and Bk using GraphPad Prism. Curves
b
shown in panels B, D, and E are the B12 representations of the curves in panels A, C, and E, respectively, using the parameters and computed
from the values of B and B0.
a
Form of the B12
Bias Functions. With the selection of
logistic response functions described above, the bias function
a
B12
is a hyperbola that can be conveniently written in one of the
three forms shown in eq II. The form (B0 + By)/(1 + y) is
used for the majority of the discussion and is defined by normalized concentration variable y, baseline B0, and asymptote B.
Parameter Bk = B B0 represents the change in the bias over
the interval y = (0,).
b
B12
(R1,
B0 + By
1
= B0 + Bk
1+y
1+y
1
= B Bk
1+y
a
(R1, R2) =
B12
y=
1+ c
1
; B =
,
1 + k1
1+
1
2( 1)
B0 =
, Bk =
1 +
(1 + )(1 + )
R2) =
1
B01 + B
y1
2
B02 + B
y2
+
1 + y1
1 + y2
a
4B12
=
a
a
(1 B12
)(1 + B12
)
a
B12
=
(prob R1)(prob R2)
c
1
1
11
1
= , B01 =
, B
, Bk1 =
;
k1
c
2
= , B02 =
y2 = , B
,
k1
1
1
1
Bk2 =
(III)
1 1
a
b
For either B12 or B12, when the response curves have equal
EC50 values (i.e., = 1), the variation parameter Bk identically
equals 0 and the bias is constant over the entire concentration
a
will change from
range. The concentration at which the bias B12
its initial value to halfway toward its final value occurs at
y1 =
(II)
b
B12
(second
Biochemistry
Article
a
Figure 2. Theoretical curves of bias parameters B and B0 as functions of the transducers. Representative curves are shown for model B12
(A and C)
b
and model B12 (B and D). Curves are parametrized by either 1/2 or = KA2/KA1.
1
2
1
B0 =
1+
1
; B =
1+
1
2
B01 =
2
B
=
1(2 + 1) 2
, B0 = 1
2(1 + 1)
2
(IVb)
1
2
+ 21
( + 1)
1 2 1
, B = 2 1
;
1
1(2 + 1)
(IVa)
Biochemistry
Article
Figure 3. Mappings of the square of bias coordinates bounded by 1 into the Poincare disk. Panels A and B depict the angle preserving
nature of the map to the disk that recapitulates the orientation and relative relationships of families of coordinate points that lie along welldefined vectors originating in the center of the square. (C) For an observer sitting on the vector at angle B in the disk, it appears that
parametrically mapped points move up or down the length of the vector to a position dependent upon the magnitude and sign of the bias. (D)
For an observer sitting on and rotating through angle Y with the mapping vector Y , it appears as if the mapped points B0 and Binf in the
Poincare disk are rotating toward the observer. Additionally, these points are projections of the vertical string that curves about the cylinder
axis and that represents the signaling response hyperbola immersed in a three-dimensional space. (E) Cartoon depicting how the string that
corresponds to the sigmoid response curve from 1 to 1 (depicted below and at the left) appears in the three-dimensional space of the
hyperbolic cylinder. The left-hand cylindrical view depicts the fixed-angle bias interpretation in which the corresponding response string is in a
frame where the mapping vector Y is not only rotating but also changing its length as it rotates (see the accompanying graph below the
cylinder at the right for the angular dependence of vector length, which is minimal at /4). Points are plotted along the fixed vector according
to the bias because of the rotation angle of the mapping vector. The right-hand view corresponds to the frame of an observer sitting on the
rotation vector that is performing the mapping. In this frame, the disks appear to rotate clockwise as the cylinder and string grow in height
with each incremental rotation.
Biochemistry
Article
Biochemistry
Article
Figure 4. Bias of compounds at the dopamine 2 receptor in different signaling paradigms. (AD) Responses are plotted for experimental dose
response data as string projections either in standard doseresponse curve format or in either a rotating or fixed-angle Poincare disk representation.
(A and B) Bias plots for -arrestin vs Gi signaling with corresponding parameter and coordinate mapping tables for the agonist quinpirole in the
absence or presence of a pathway enhancer (GRK) or inhibitor (pertussis toxin). Distances, , from the corresponding quinpirole point are provided
in the table for nonrotated and rotated coordinates. To provide a comparison of two strategies for calculating bias curves, curves for quinpirole have
been calculated from (1) fitting the individual Gi and -arrestin responses, separately determining the individual Pi and Ki, and then calculating the
bias curve (orange curve) or (2) directly fitting the two response ratios (g f)/(g + f) at different concentrations [, 95% confidence interval (see
the eighth section of Theoretical Calculations)]. Additionally, the first and last coordinates plotted for the quinpirole + GRK curve (green) are
bounded by closed curves defining neighborhoods of points within a distance of 0.22 [m = 0.32 (see the sixth section of Theoretical Calculations)],
or with a <25% difference in the g/f ratio of the bias curves. (C) Plots of the bias of quinpirole and terguride for -arrestin signaling in the absence
and presence of added GRK, a -arrestin pathway enhancer. (D) Plots representing the bias of different D2 receptor antagonists for inhibiting either
the -arrestin pathway or Gi signaling.
DISCUSSION
The recent recognition that altering a receptors conformational
space has physiological consequences has accelerated searches
for biased compounds. This study addresses a lack of formalism
in receptor bias analysis by characterizing the mathematical
relationship between signaling bias and the hyperbolae that
commonly describe biological responses. Specifically, we
incorporate the response functions into an axiomatic system
that defines signaling bias rather than determining the nature of
bias on the basis of the responses. Our study shows that (1)
coordinates (B0,B) of paired bias coefficients form a unit
square indexing response hyperbolae, (2) the signaling
response hyperbolae form groups of families defined by
direction vectors centered about (0,0) in the unit square, (3)
Biochemistry
ACKNOWLEDGMENTS
We thank Professor Marc Caron for stimulating discussions
concerning receptor bias and its significance to pharmacology
and drug innovation.
Article
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