37.L.M.Palmer,G.J.Stuart,J. Neurosci. 29 , 6897– 6903
(2009).
- A. M. Packeret al.,Nat. Methods 9 , 1202–1205 (2012).
- N. C. Klapoetkeet al.,Nat. Methods 11 , 338–346 (2014).
- C. Koch, A. Zador,J. Neurosci. 13 , 413–422 (1993).
- R. Yuste, M. J. Gutnick, D. Saar, K. D. Delaney, D. W. Tank,
Neuron 13 , 23–43 (1994). - J. Schiller, Y. Schiller, G. Stuart, B. Sakmann,J. Physiol. 505 ,
605 – 616 (1997). - K. Holthoff, Y. Kovalchuk, R. Yuste, A. Konnerth,J. Physiol.
560 , 27–36 (2004). - H. Jia, N. L. Rochefort, X. Chen, A. Konnerth,Nature 464 ,
1307 – 1312 (2010). - T. W. Chenet al.,Nature 499 , 295–300 (2013).
- D. E. Wilson, D. E. Whitney, B. Scholl, D. Fitzpatrick,
Nat. Neurosci. 19 , 1003–1009 (2016). - I. Segev, W. Rall,J. Neurophysiol. 60 , 499–523 (1988).
- R. Araya, V. Nikolenko, K. B. Eisenthal, R. Yuste,Proc. Natl.
Acad. Sci. U.S.A. 104 , 12347–12352 (2007). - N. L. Golding, N. Spruston,Neuron 21 , 1189–1200 (1998).
- D. Holcman, Z. Schuss,J. Math. Neurosci. 1 , 10 (2011).
51. T. Lagache, K. Jayant, R. Yuste,J. Comput. Neurosci. 47 ,
77 – 89 (2019).
52. R. Yuste,Neuron 71 , 772–781 (2011).
53. W. Rall, inStudies in Neurophysiology, R. Porter, Ed.
(Cambridge Univ. Press, 1974), pp. 203–209.
54. M. London, M. Häusser,Annu. Rev. Neurosci. 28 , 503– 532
(2005).
55. D. P. Purpura,Science 186 , 1126–1128 (1974).
ACKNOWLEDGMENTS
We thank W. Stoy for comments and data in Fig. 1C and fig. S2C.
R.Y. is an Ikerbasque Research Professor at the Donostia
International Physics Center. This work is dedicated to the memory
of Amiram Grinvald.Funding:This work was funded by National
Institute of Neurological Disorders and Stroke (NINDS) grant
R01NS110422 (R.Y.), NINDS grant R34NS116740 (R.Y.), National
Eye Institute (NEI) grant R01EY011787 (R.Y.), National Institute of
Mental Health (NIMH) grant R01MH115900 (R.Y.), and the PEW
Latin American Fellows Program in Biomedical Sciences (V.H.C.).
Author contributions:Conceptualization: V.H.C., R.Y.; Methodology:
V.H.C., R.Y.; Software: V.H.C., N.O.; Formal analysis: V.H.C., N.O.;Investigation: V.H.C.; Writing–original draft: V.H.C., R.Y.; Writing–
review and editing: V.H.C., N.O., R.Y.; Visualization: V.H.C.;
Supervision: R.Y.; Project administration: R.Y.; Funding acquisition:
R.Y.Competing interests:The authors declare no competing
interests.Data and materials availability:All data are available in
the manuscript and the supplementary materials.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abg0501
Materials and Methods
Supplementary Text
Figs. S1 to S12
References ( 56 – 64 )
MDAR Reproducibility Checklist
Movies S1 and S211 December 2020; accepted 1 November 2021
Published online 11 November 2021
10.1126/science.abg0501STRUCTURAL BIOLOGY
Cryo-EM structure of human GPR158 receptor
coupled to the RGS7-Gb5 signaling complex
Dipak N. Patil^1 †, Shikha Singh^2 †, Thibaut Laboute^1 , Timothy S. Strutzenberg^3 , Xingyu Qiu4,5, Di Wu4,5,
Scott J. Novick^3 , Carol V. Robinson4,5, Patrick R. Griffin^3 , John F. Hunt^2 , Tina Izard^6 ,
Appu K. Singh7,8, Kirill A. Martemyanov^1
GPR158 is an orphan G protein–coupled receptor (GPCR) highly expressed in the brain, where it controls
synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and
cognition. However, the structural organization and signaling mechanisms of GPR158 are largely
unknown. We used single-particle cryo–electron microscopy (cryo-EM) to determine the structures of
human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric
organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an
unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158
coupling to RGS7-Gb5. Together, these results provide insights into the unusual biology of orphan
receptors and the formation of GPCR-RGS complexes.
G
protein–coupled receptors (GPCRs),
which form the largest family of pro-
teins encoded in mammalian genomes,
detect extracellular signals to program
cellular response. GPCRs are essential
to understanding physiology, disease, and drug
development ( 1 , 2 ). The canonical model posits
that GPCRs transduce their signals by recruit-
ment and activation of heterotrimeric G pro-
teins ( 3 ). This model was subsequently updated
to accommodate alternative signal propaga-
tion by recruitment ofb-arrestin scaffolds ( 4 ).
Termination of GPCR signaling requires the
action of RGS (regulator of G protein signaling)
proteins, which directly deactivate G proteins
( 5 , 6 ). GPCRs and RGS are thus classically
considered as opposing forces in controlling
cellular responses. However, they have long
been reported to form complexes, which sug-
gests the existence of additional signaling
mechanisms ( 7 , 8 ).
Orphan GPCRs are attractive drug targets
with important roles in physiology and disease
( 9 , 10 ). Yet in many cases, their mechanisms,
ligands, and signaling reactions are poorly
understood. An example is the orphan recep-
tor GPR158. It is one of the most abundant
GPCRs in the brain, well documented for its
pivotal role in regulating mood and cognition,
andisimplicatedinarangeofdiseases( 11 – 15 ).
It shapes synaptic organization and function
by regulating ion channels and second mes-
sengers ( 16 , 17 ). GPR158 features a large ex-tracellular domain with distinctive sequence
suggesting unique ligand recognition prin-
ciples. The central feature of GPR158 is its
association with the neuronal RGS7-Gb5 pro-
tein complex ( 11 ). Binding with GPR158 poten-
tiates RGS activity ( 18 ), and both proteins
act together to regulate homeostasis of the
second messenger cyclic adenosine mono-
phosphate (cAMP) to control neuronal activity
with marked impact on brain physiology ( 19 ).
However, its signaling mechanisms and struc-
tural organization remain elusive.
We used single-particle cryo–electron mi-
croscopy (cryo-EM) to obtain structures of
GPR158 in the apo state and in complex with
RGS7-Gb5 at an average resolution of 3.4 Å
and 3.3 Å, respectively (figs. S1 to S3 and table
S1). The structure of GPR158 reveals a homo-
dimer assembly (Fig. 1A) where the dimeriza-
tion interface involves the extracellular domain
(ECD), the transmembrane (TM) region, and
cytoplasmic elements (Fig. 1A and fig. S4A).
Each protomer features prominent extra-
cellular and transmembrane domains linked
by a flexible“stalk”domain. The N-terminal
portion of the ECD adopts a characteristic
Cache domain fold (the name derives from
“calcium channels and chemotaxis recep-
tors”). The TM region of protomers contains
well-resolved helices. We observed continu-
ous density for TM1 through TM7 including
all extra- and intracellular loops (ECLs and
ICLs), except for ICL2. We further detected
two phospholipids in the cavity generated by
the dimeric interface and several cholesterol-
like molecules packed against hydrophobic
residues of the TM domain, including the
dimeric interface. The density at the ECD is
limited, and many of the side-chain densities
are not visible. The overallBfactor and aver-
age side-chainBfactor for ectodomain are
high (>50 Å) (fig. S4C), suggesting greater
conformational flexibility of the ECD. None-
theless, the key organizational features of ECD
are clearly distinguishable.86 7 JANUARY 2022•VOL 375 ISSUE 6576 science.orgSCIENCE
(^1) Department of Neuroscience, The Scripps Research
Institute, Jupiter, FL 33458, USA.^2 Department of Biological
Sciences, Columbia University, New York, NY 10027, USA.
(^3) Department of Molecular Medicine, The Scripps Research
Institute, Jupiter, FL 33458, USA.^4 Department of Chemistry,
University of Oxford, Oxford OX1 3TA, UK.^5 Kavli Institute for
Nanoscience Discovery, Oxford OX1 3QU, UK.^6 Department
of Integrative Structural and Computational Biology, The
Scripps Research Institute, Jupiter, FL 33458, USA.
(^7) Department of Biological Sciences and Bioengineering,
Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh
208016, India.^8 Mehta Family Centre for Engineering in
Medicine, Indian Institute of Technology Kanpur, Kanpur,
Uttar Pradesh 208016, India.
*Corresponding author. Email: [email protected] (K.A.M.);
[email protected] (A.K.S.)
†These authors contributed equally to this work.
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