Abstract
A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.
Highlights
A GPCRs2 play a critical role in normal cell function and are the focus of intense studies and targets for drug development
Detection of Agonist-induced Conformational Changes in the N Terminus of -Opioid Receptors—Studies with rhodopsin indicate that the N-terminal region undergoes conformational changes following receptor activation
The involvement of the transmembrane regions in conformational changes following the binding of the agonist to the receptor has been extensively documented [1,2,3,4,5,6,7,8,9,10], not much is known about the involvement of the N-terminal region
Summary
Cell Culture and Transfection—CHO cells stably expressing FLAG-tagged mouse receptors were grown in F-12 medium [23]. Cells were quickly rinsed three times (within 5 min) with cold PBS (washing with 20 M antagonist in PBS produced similar results) and fixed with ice-cold methanol for 10 min at Ϫ20 °C This treatment was included to help reduce cell loss during multiple washings as determined by protein estimation or recognition by FLAG Ab. We do not observe any significant differences in receptor recognition by SA25 antiserum or FLAG monoclonal Ab in CHO- cells that were subjected or not to methanol fixation (0.29 Ϯ 0.01 without and 0.35 Ϯ 0.04 with methanol fixation for SA25 Ab and 0.22 Ϯ 0.01 without and 0.23 Ϯ 0.01 with methanol fixation for FLAG Ab). To examine the effect of modulators of G-protein activity, cortical membranes (10 g) were pretreated with a 100 M concentration of either GTP␥S, GPP(NH)p, AlF3, or NaF for 30 min at 37 °C in the presence of a protease inhibitor mixture (Sigma) This was followed by a 30-min treatment with 1 M DAMGO, and the extent of Ab recognition was assayed by ELISA as described above. The reaction product was transferred to a 96-well plate, and absorbance at 490 nm was measured with a Bio-Rad ELISA reader
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