Molecular Recognition Section, Division of Intramural Research : NIDDK : National Institutes of Health
NavigationINDEX

Molecular Biology


On this page, we have put some data together abstracted from previous and current work in our lab. We are still working on most of these studies, and the tables will be updated as more data become available.

Part of this work was published in:

We also have an analysis of the molecular modelling aspects of this work on-line.

Before venturing into site-directed mutagenesis experiments, we devised a strategy to detect receptors that lacked binding of radioligands. This would enable us to distinguish between (greatly) reduced affinity of the mutant receptors for the radioligands, and reduced expression levels of the mutant protein at the cell-surface.

Thus, an immunologically detectable HA-tag (hemagglutinin) was inserted into the human A2A adenosine receptor sequence ( Table I ) immediately following the first Met residue. The resulting Tag1 receptor showed virtually the same affinity for [3H]CGS 21680 as the wild type receptor ( Table II), but could not be detected by ELISA, Western blotting, or immunocytochemistry.

Table I: N-terminal sequences of epitope-tagged human A2A receptors.
wild type human A2A sequence MPIMGSS..........
Tag1 sequence MYPYDVPDYAPIMGSS..........
Tag2 sequence MPIMYPYDVPDYAGSS..........
Tag3 sequence PIMYPYDVPDYAGSS..........

Interestingly, there are two methionine residues in the amino-terminal segment of the human A2A adenosine receptor. The introduction of an HA-tag following the first Met might affect the initiation of protein translation, causing protein expression to start at the second Met. This would result in the deletion of the first 3 amino acids (MPI) and the HA-tag not being translated. However, the resulting mutant receptor may still be functional. We therefore introduced an HA-tag after the second Met ( Tag2 ). This mutant receptor showed the same affinity for [3H]CGS 21680 and the same expression level (Bmax) as the non-tagged receptor( Table II ). Moreover, the Tag2 receptor could also be detected by ELISA (but not Western blotting).

Table II: [3H]CGS 21680 binding properties of wild type (WT) and three epitope-tagged human A2A adenosine receptor constructs.
construct Kd
nM
Bmax
pmol/mg
A2A WT 15.9 ± 0.3 26.9 ± 13.3
Tag1 24.1 ± 0.1 24.3 ± 12.4
Tag2 23.8 ± 2.0 28.0 ± 14.4
Tag3 21.7 ± 2.6 35.4 ± 20.0

To test if the first Met is important for the expression of the receptor, we deleted the first Met to construct Tag3. Again, the Tag3 receptor displayed affinity and expression levels similar to the wild type receptor ( Table II ). We therefore concluded that the second Met may represent the physiological translation start site in the human A2A adenosine receptor. Based on these results, the Tag3 epitope-tag was used in further site-directed mutagenesis studies.

First off, we tried to localize residues involved in ligand binding present in the fifth, sixth and seventh transmembrane domains (TM5, TM6 & TM7; Table III ).

Table III: Effects on [3H]CGS 21680 binding to wild type (WT-Tag3) and mutant human A2A receptors.
Kd/i (nM) CGS 21680 CADO NECA R-PIA CGS 15943 XAC
WT (Tag3) 21.7 ± 2.6 152 ± 10 19.9 ± 8.3 318 ± 8.0 10.0 ± 2.0 63.2 ± 5.5
F180A 15.1 ± 1.3 ND 46.0 ± 11.1 ND 13.7 ± 3.2 90.3 ± 7.2
N181S 24.8 ± 5.6 1340 ± 200 16.4 ± 2.5 4500 ± 1200 23.7 ± 2.0 130 ± 10.0
F182Y 57 ± 15 1890 ± 170 170 ± 11 2800 ± 1100 19.2 ± 4.2 140 ± 20.1
F182W 66 ± 6 776 ± 120 144 ± 12 1600 ± 460 10.3 ± 3.3 66.8 ± 1.0
H250Y 14.9 ± 1.0 8250 ± 1400 43.8 ± 16 9580 ± 280 8.4 ± 1.0 52.7 ± 11
H250F 14.4 ± 4.0 2130 ± 100 41.9 ± 12 7950 ± 1750 25.7 ± 5.0 60.3 ± 3.0
C254A 23.7 ± 2.4 ND 18.2 ± 10.3 ND 19.4 ± 3.4 54.1 ± 8.2
S281T 17.1 ± 2.0 13.7 ± 1.3 6.6 ± 0.8 30.5 ± 8.6 15.6 ± 3.4 17.3 ± 1.6

In general, these mutations did not affect the affinity of antagonists (such as CGS 15943 and XAC), but the effects on agonist (CGS 21680, CADO, NECA, and R-PIA) binding were more intricate.

The F180A, and C254A mutant receptors showed ligand binding properties similar to those of the wild type receptor. Therefore, the aromatic group in F180, and the thiol group in C254 are not required for binding of agonists or antagonists.

A detailed analysis of these results is provided in our paper cited above.

A particular residue in TM7 (S277) had some very interesting features. We decided to have a closer look, and measured both [3H]CGS 21680 binding when possible and [3H]XAC binding when [3H]CGS 21680 binding was virtually absent ( Table IV ).

Table IV: Radioligand binding to human A2A receptors.
Kd/i (nM) CGS 21680 CADO NECA R-PIA CGS 15943 XAC
[3H]CGS 21680
WT (Tag3) 21.7 ± 2.6 152 ± 10 19.9 ± 8.3 318 ± 8.0 10.0 ± 2.0 63.2 ± 5.5
S277A < 2 % SB
S277N 25.0 ± 5.0 221 ± 21 43.0 ± 17.1 631 ± 329 12.8 ± 2.8 60.0 ± 17.4
S277T 23.6 ± 6.3 201 ± 5.0 39.8 ± 6.9 534 ± 152 12.2 ± 1.0 65.9 ± 2.2
S277C 37.2 ± 1.2 384 ± 22 129 ± 12 1588 ± 570 1.53 ± 1.1 10.3 ± 4.6
[3H]XAC
WT (Tag3) 40.7 ± 9.5 208 ± 66.4 72.5 ± 10.0 792 ± 49.9 20.7 ± 0.8 9.4 ± 2.3
S277A ~ 43600 ~ 81300 ~ 29100 ~ 33800 17.5 ± 4.1 6.7 ± 1.2

Some mutants simply prevented ligand binding at all, regardless of whether the receptor was expressed at the cell surface (measured by immunological detection of the Tag3 sequence), or whether the (mutant) receptor was present and could still activate adenylyl cyclase ( Table V ).

Table V: Detrimental Mutations to human A2A receptors.
mutant(s) [3H]CGS 21680 [3H]XAC expression
level (%)
adenylyl cyclase
(EC50; nM)
WT (Tag3) + + 100 3.0 ± 1.0
F182A - - 71 ± 13
H250A - - 58 ± 1
N253A,Q,S - -,-,- 40 ± 6 (A)
F257A - - 68 ± 16
I274A - - 72 ± 15 100 ± 20
S277A - + 25 ± 6 210 ± 35
H278A,F,Q,Y - -,-,-,- 72 ± 24 (A) 920 ± 70 (A)
S281A - - 36 ± 7

If you would like to know more about

you have found the right spot:
MRS Home Page
Staff
Selected Publications
Adenosine Receptors
P2 Receptors
Medicinal Chemistry
Computational Chemistry
The Purine Club
GPCRs
Links

Chief: Dr. Kenneth Jacobson
The Molecular Recognition Section (MRS) is in the Laboratory of Bioorganic Chemistry at the National Institute of Diabetes and Digestive and Kidney Diseases which is part of the National Institutes of Health, Bethesda, MD, USA. General inquiries may be addressed to NIDDK, NIH, Building 31, Room 9A04, 31 Center Drive, MSC 2560, Bethesda, MD 20892-2560, USA.
Privacy || Copyright, Disclaimers and Access Restrictions || Accessibility

National Institute of Diabetes, Digestive & Kidney Diseases U. S. National Institutes of Health U.S. Department of Health & Human Services USA.gov Logo - link to the U.S. government's official web portal
National Institutes of Health