MCAT practice · Biological and Biochemical Foundations · 3A Nervous and endocrine systems · 1A Proteins and enzymes
Agonists, an antagonist and desensitization at a Gs-coupled receptor
An original practice passage written for this site, not an AAMC item. Exam pace is about 8 minutes for a passage like this.
The β2-adrenergic receptor is a G protein-coupled receptor that signals through Gαs to adenylyl cyclase. Researchers characterized three ligands in a line of cultured kidney cells that stably expresses the human receptor: agonist A, compound B and compound P.
In Experiment 1, cells were incubated for 15 minutes with increasing concentrations of agonist A or compound B, and intracellular cAMP was measured by immunoassay. Every incubation contained IBMX, an inhibitor of cyclic nucleotide phosphodiesterases. To test compound P, cells were exposed to 10 nM compound P for 10 minutes before agonist A was added. Responses were expressed as a percentage of the maximal cAMP rise produced by agonist A alone (Figure 1). Agonist A had an EC50 of about 3 nM. In the same cells, phosphorylation of CREB, a transcription factor that is a substrate of protein kinase A, reached half its maximum at about 0.3 nM agonist A.
In Experiment 2, the researchers asked why responses faded during prolonged exposure. Cells were pretreated for 30 minutes with vehicle, with 1 µM agonist A, or with 1 µM agonist A plus compound G, a selective inhibitor of G protein-coupled receptor kinase 2 (GRK2). The cells were washed three times, rested for 5 minutes, and then challenged for 10 minutes with either 1 µM agonist A or 10 µM forskolin, a plant diterpene that binds and activates adenylyl cyclase directly. Basal cAMP in unstimulated cells was about 4 pmol per well under all three pretreatments (Table 1).
Pretreatment with agonist A cut the response to a second dose of agonist A by about two thirds, while the forskolin response changed little. Compound G largely prevented the loss. The researchers concluded that prolonged agonist exposure weakens the receptor's coupling to the cascade.
- Agonist A
- Agonist A + 10 nM compound P
- Compound B
| Pretreatment | Challenge: 1 µM agonist A (pmol/well) | Challenge: 10 µM forskolin (pmol/well) |
|---|---|---|
| Vehicle | 52 ± 3 | 121 ± 6 |
| 1 µM agonist A | 18 ± 2 | 114 ± 7 |
| 1 µM agonist A + compound G | 44 ± 3 | 118 ± 5 |
Data are illustrative, constructed for practice.
1. Which statement best describes the effect of compound P in Figure 1 and the kind of antagonism it suggests?
- A. Wrong: the EC50 did rise, but the ceiling stayed near 100%. A maximum near 45% is the compound B curve, which belongs to a different ligand.
- B. Right: the half-maximal point moves from about 3 nM (log −8.5) to about 30 nM (log −7.5) and the plateau is unchanged. A rightward shift with the same ceiling means enough agonist outcompetes P for the same site.
- C. Wrong: the curve with compound P still reaches about 100% at high agonist A, so the maximum is not lowered; the curve moves sideways, not down.
- D. Wrong: this is a direction error. The curve moved to the right, toward higher concentrations, so more agonist is needed and the EC50 rose rather than fell.
2. In Experiment 2, what was the main purpose of challenging some pretreated cells with forskolin rather than with agonist A?
- A. Wrong: forskolin acts on adenylyl cyclase, not on the receptor, so a forskolin response says nothing about where compound G binds. Compound G is described as a GRK2 inhibitor.
- B. Wrong: forskolin is not a receptor agonist. It bypasses the receptor and the G protein, which is exactly why it is useful here.
- C. Right: forskolin activates adenylyl cyclase directly. Its response was nearly unchanged after pretreatment (121 versus 114 pmol), so the cyclase and cAMP measurement were intact and the loss lies upstream, at the receptor or its coupling to Gs.
- D. Wrong: blocking phosphodiesterase is what IBMX did in Experiment 1. Forskolin raises cAMP by activating its synthesis, not by slowing its breakdown.
3. Cholera toxin ADP-ribosylates Gαs. If cells like those in Experiment 1 were first treated with cholera toxin, what would basal cAMP (no agonist) most likely be, and how would adding compound P alone affect it?
- A. Wrong: this reverses the toxin's effect. Cholera toxin prevents GTP hydrolysis rather than GTP binding, so Gαs is stuck in the active state and cAMP rises.
- B. Wrong: cholera toxin modifies the G protein, not the receptor, and it raises cAMP. A neutral antagonist added alone also has no agonist response to block.
- C. Wrong: the first half is right, but the toxin locks Gαs on whether or not the receptor is active. Removing the receptor's stimulus does not turn off a G protein that cannot hydrolyze GTP.
- D. Right: ADP-ribosylation blocks the intrinsic GTPase of Gαs, so Gαs stays GTP-bound and keeps adenylyl cyclase on with no receptor input. An antagonist acts at the receptor, upstream of the lesion, so it cannot reverse the rise.
4. Agonist A half-maximally stimulated CREB phosphorylation at about one tenth the concentration needed to half-maximally raise cAMP. Which explanation is most consistent with this finding?
- A. Wrong: agonist A acts through the cell-surface receptor, and protein kinase A is activated by cAMP, not by the agonist itself. Nothing in the passage suggests a second, intracellular site.
- B. Right: each enzyme step multiplies the signal, so the downstream output reaches its maximum when cAMP is only partly raised. Downstream readouts therefore show a lower EC50 than upstream ones.
- C. Wrong: desensitization weakens the response, which would move the curve to the right and raise the EC50, the opposite of what was seen.
- D. Wrong: all the responses come from one receptor population coupled to Gs, and the passage links CREB to the receptor only through cAMP and protein kinase A. EC50 differences between readouts here reflect the cascade, not receptor subtypes.
5. Cells are incubated with 1 µM agonist A and 100 µM compound B added together. Assuming both ligands bind the same site on the receptor with similar affinity, cAMP accumulation would most likely:
- A. Right: B is a partial agonist (its ceiling is about 45% in Figure 1). At a hundred times the concentration of A and with similar affinity, B occupies about 99% of the receptors, so the response falls toward B's own maximum. A partial agonist acts as an antagonist against a full agonist.
- B. Wrong: being an agonist is not the same as having equal efficacy. Figure 1 shows B's maximum is less than half of A's, so replacing A with B lowers the response.
- C. Wrong: two ligands competing for one site do not add their effects. At most each receptor is occupied by one ligand, and B produces less activation per receptor than A.
- D. Wrong: B is not a silent antagonist; it raises cAMP on its own to about 45%. Displacing A with B reduces the response but does not abolish it.
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