After doing a whole bunch of cutting-edge papers for the class its time to go back in time a bit (like 1998 is ancient but anyways) and do an oldie-but-goodie. This particular paper, “Effector Pathway-Dependent Relative Efficacy at Serotonin Type 2A and 2C Receptors: Evidence for Agonist-Directed Trafficking of Receptor Stimulus”, Berg et al., 1998 Molecular Pharmacology (Free at Mol Pharm) isn’t really a citation classic (with 278 citations according to google scholar), yet, it marks a very significant moment in GPCR pharmacology. I like this paper for two reasons: 1) It moved a major, emerging pharmacology theoretical framework forward toward experimental discovery and 2) I am very fond of the first and last authors.
First to my fondness for the first and last authors, Kelly Berg and Bill Clarke. Bill and Kelly are professors in the Department of Pharmacology at The University of Texas Health Science Center at San Antonio (UTHSCSA). It so happens that I did my PhD in that very department (I started there in 1998). The very first class I took was Bill Clarke’s Principles of Pharmacology course. When I joined the department I was quite sure I wanted to be a pharmacologist but this course drove that point home for me in ways that are difficult to describe. The course was mainly taught by Bill and Kelly (who happen to be married) with Bill doing most of the teaching on basic principles and Kelly doing the teaching on molecular signaling through GPCRs. While I learned an enormous amount about basic pharmacological principles and the ins-and-outs of GPCR signaling in the class my main memories are of the passion for teaching and graduate education that they both passed on to all of us throughout the semester. I like to think that my teaching style came mostly from the two of them and while I am sure I have not yet lived up to their level of excellence, their example consistently gives me a goal to shoot for. In this class I like to use this paper to transition from screening technologies back to pharmacological principles largely because it reminds me to try to live up to what BIll and Kelly imparted to me through their course.
Okay, enough nostalgia, onto the paper…
Figure 1 tells you more or less all you need to know.
The idea is very simple: can different agonists cause differential activation of signaling pathways through agonist action at the same receptor? The diagram shows three agonists at a single receptor. Agonist 1 (A1) preferentially activates effector pathway 1 (E1). A2 activates both pathways equally and A3 preferentially activates E2. Together, this diagram demonstrates agonist-directed trafficking of receptor stimulus (which we now tend to just call agonist-directed trafficking). Presumably, agonist 1 pushes the receptor toward a conformation that prefers activation of pathway E1 while A3 pushes toward another conformation that prefers pathway E2. A2 either stabilizes both of these conformations equally or pushes the receptor toward a third conformation that equally stimulates both pathways. This is a well-accepted premise now but at the time of publication it was just an idea floating around suggested by GPCR theory and some findings that suggested such effects but no one had ever nailed it down convincingly:
Recently, there have been several reports of differential effector activation by agonists that are difficult to explain with traditional receptor theory (see Kenakin, 1995, 1996, and references therein). For example, Spengler et al. (1993) found a reversal of the potency of two agonists to elicit cAMP accumulation and PLC-mediated IP accumulation by activating the PACAP receptor transfected transiently into LLC PK1 cells. PACAP1–27 had a slightly greater potency than PACAP1–38 for cAMP accumulation, whereas PACAP1–38 was considerably more potent for IP accumulation than PACAP1–27. Because the affinity of each of these agonists for the PACAP receptor is the same regardless of the response measured, the differences in potency must be the result of differences in the efficacy of the agonists to elicit each response. Robb et al. (1994) reported differences in the potency for octopamine and tyramine to inhibit cAMP accumulation and to increase [Ca2+]i by activating theDrosophila melanogaster octopamine-tyramine receptor expressed stably in CHO cells. Tyramine was almost 2 orders of magnitude more potent than octopamine in inhibiting cAMP accumulation, whereas octopamine was more potent than tyramine when the kinetics (but not the magnitude) of changes in [Ca2+]i were measured.
As often happens in GPCR pharmacology, Terry Kenakin stepped in proposed a theoretical framework to explain these findings. If you are not familiar with the work of Terry Kenakin I suggest you head over to pubmed and check out some of his more recent drug discovery and GPCR reviews. They are fabulous. So what did he propose?
To help explain these experimental observations, Kenakin proposed a new concept of agonist action, termed “agonist-directed trafficking of receptor stimulus,” in which agonists can preferentially induce/select receptor conformational states that favor activation of one effector pathway over another (Kenakin, 1995). Computational simulations of ligand interactions with the 5-HT2A receptor (Zhang and Weinstein, 1993) and recent experimental evidence with the β2-adrenergic receptor (Gether et al., 1995; Krumins and Barber, 1997) support the concept of agonist-selective receptor states, although there is some debate as to the number of receptor conformational states (Leff et al., 1997). For a receptor that couples to multiple signal transduction pathways within a cell, one consequence of agonist-selective receptor states can be differential activation of effectors (Fig.1A). Thus, in contrast to traditional receptor theory, agonist-directed trafficking hypothesis predicts that the efficiency of receptor coupling to each of multiple effector pathways in a cell is a function of the agonist, and consequently agonist relative efficacy could be effector pathway dependent (Fig.1B).
But there was a problem:
Although there is some experimental support for agonist-directed trafficking (Spengler et al., 1993; Robb et al., 1994), the hypothesis has not been subjected to rigorous testing. Because acceptance of the concept that agonists can differentially activate effector pathways coupled to the same receptor will have a considerable impact in many areas of pharmacology, physiology, and therapeutics (see Results and Discussion), it is vital that rigorous testing be done.
5-HT2A and 5-HT2C receptors couple to PLC-IP and PLA2-AA pathways in brain tissue (Felder et al., 1990; Kaufman et al., 1995) and in heterologous expression systems (Berg et al., 1994b, 1996). To test the agonist-directed trafficking hypothesis under carefully controlled experimental conditions, we measured the relative efficacies of a series of 5-HT2A/2C receptor agonists on each of the two effector pathways coupled to human 5-HT2A and 5-HT2C receptors expressed stably in CHO cells. We also examined the capacity of the three-state model to accommodate the experimental data.
The point of the paper, then, is to show that agonist-directed trafficking of receptor stimulus can occur. To do this they used CHO cells heterologously expressing either the 5HT2A or 5HT2C receptors. Agonists for either of these receptors failed to activate signaling in CHO cells not transfected with 5HT2A or 5HT2C and when the receptors were expressed there was no observation of receptor reserve. This was an important observation because the presence of receptor reserve can change the observed intrinsic efficacy of a given agonist. They showed the absence of receptor reserve by using an irreversible (non-competitive) antagonist of 5HT2A receptors. In the presence of a full agonist (5HT) the irreversible antagonist downshifted the Emax for the agonist without changing to EC50. If there was receptor reserve in the system they would have observed a right-shift in the EC50 and possibly a down-shift in the Emax (depending on the concentration of the irreversible antagonist — higher concentrations would give a right shift and a decrease in Emax). Competitive antagonists only shift the EC50 to the right and generally have no effect on the Emax because an agonist can always overcome the antagonist (definition of competitive). Competitive antagonists are generally not helpful in determining the presence of receptor reserve.
To look at differential activation of downstream effectors in their system they measures IP accumulation and arachidonic acid release. IP accumulation is linked to phospholipase C (PLC) activity and AA release is linked to phospholipase A2 (PLA2) activity. These different signaling pathways were measured simultaneously in cells exposed to different 5HT2A/2C agonists. The prediction, if agonist-directed trafficking occurs, is that different agonists would preferentially stimulate one pathway vs. the other in CHO cells. Figure 4 shows that the data fits the prediction:
DOI and LSD preferentially activate the AA pathway while TFMPP and quipazine preferentially activate the IP pathways. Now this demonstration is not perfect as we would ultimately like to see an agonist at 5HT2C (in this case) that only stimulates the AA or IP pathway and has no effect on the other pathway; however, the data still demonstrates that some preferential activation of downstream effectors can be achieved with specific agonists of a given receptor. Data in Fig 5 shows that this can also occurs with agonists for 5HT2A receptors. Finally, the authors use a three-state model for receptor coupling to fit the observed data. Equations for the three-state model are shown in the methods. They show that this three-state model fits the observed data very well, as would be expected. Hence, from these findings, we can conclude that agonist-directed trafficking of receptor stimulus can occur, at least for 5HT2A/C receptors in a reconstituted system (CHO cells). To my knowledge, this was the first definitive demonstration of agonist directed trafficking.
This paper is quite old in terms of progress in pharmacology theory as it pertains to GPCR agonism and effector pathway stimulation. The field has expanded dramatically since this landmark paper and we have now moved far beyond this relatively simple three state model into much more complex probability based models of agonist biasing of receptor conformations and effector pathway stimulation. I don’t have time to go into this in more detail but if you are interested I highly suggest this paper from Terry Kenakin for further reading.
Berg KA, Maayani S, Goldfarb J, Scaramellini C, Leff P, & Clarke WP (1998). Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Molecular pharmacology, 54 (1), 94-104 PMID: 9658194