A new class of receptors: Lipids regulate mammalian Gsα-stimulated adenylyl cyclase activities via their membrane anchors

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Weaknesses:

(1) Important methodological details regarding the treatment of mAC membrane preps with fatty acids are missing.

(2) It is not evident that fatty acid regulators can be considered as "signaling molecules" since it is not clear (at least to this reviewer) how concentrations of free fatty acids in plasma or endocytic membranes are hormonally or otherwise regulated.

Reviewer #2 (Public review):

Summary:

The authors extend their earlier findings with bacterial adenylyl cyclases to mammalian enzymes. They show that certain aliphatic lipids activate adenylyl cyclases in the absence of stimulatory G proteins and that lipids can modulate activation by G proteins. Adding lipids to cells expressing specific isoforms of adenylyl cyclases could regulate cAMP production, suggesting that adenylyl cyclases could serve as 'receptors'.

Strengths:

This is the first report of lipids regulating mammalian adenylyl cyclases directly. The evidence is based on biochemical assays with purified proteins, or in cells expressing specific isoforms of adenylyl cyclases.

Weaknesses:

It is not clear if the concentrations of lipids used in assays are physiologically relevant. Nor is there evidence to show that the specific lipids that activate or inhibit adenylyl cyclases are present at the concentrations required in cell membranes. Nor is there any evidence to indicate that this method of regulation is seen in cells under relevant stimuli.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Weaknesses:

(1) At the beginning of the results section, the authors say "We have expected lipids as ligands". It is not quite clear why these could not have been other substances. It is because they were expected to bind in the lipophilic membrane anchors? Various lipophilic and hydrophilic ligands are known for GPCR which also have transmembrane domains. Maybe 1-2 additional sentences could be helpful here.

(2) In stably transfected HEK cells expressing mAC3 or mAC5, they have used only one dose of isoproterenol (2.5 uM) for submaximal AC activation. The reference 28 provided here (PMID: 33208818) did not specifically look at Iso and endogenous beta2 adrenergic receptors expressed in HEK cells. As far as I remember from the old pharmacological literature, this concentration is indeed submaximal in receptor binding assays but regarding AC activity and cAMP generation (which happen after signal amplification with a so-called receptor reserve), lower Iso amounts would be submaximal. When we measure cAMP, these are rather 10 to 100 nM but no more than 1 uM at which concentration response dependencies usually saturate. Have the authors tried lower Iso concentrations to prestimulate intracellular cAMP formation? I am asking this because, with lower Iso prestimulation, the subsequent stimulatory effects of AC ligands could be even greater.

(3) The authors refer to HEK cell models as "in vivo". I agree that these are intact cells and an important model to start with. It would be very nice to see the effects of the new ligands in other physiologically relevant types of cells, and how they modulate cAMP production under even more physiological conditions. Probably, this is a topic for follow-up studies.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The authors have achieved their aims to a very high degree, their results do nicely support their conclusions. There is only one point (various classical GPCR concentrations, please see above) that would be beneficial to address.

Without any doubt, this is a groundbreaking study that will have profound implications in the field for the next years/decades. Since it is now clear that mammalian adenylyl cyclases are receptors for aliphatic fatty acids and anandamide, this will change our view on the whole signaling pathway and initiate many new studies looking at the biological function and pathophysiological implications of this mechanism. The manuscript is outstanding.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Weaknesses:

(1) Important methodological details regarding the treatment of mAC membrane preps with fatty acids are missing.

We will address this issue in more detail.

(2) It is not evident that fatty acid regulators can be considered as "signaling molecules" since it is not clear (at least to this reviewer) how concentrations of free fatty acids in plasma or endocytic membranes are hormonally or otherwise regulated.

Although this question is not the subject of this ms., we will address this question in more detail in the discussion of the revision.

Reviewer #2 (Public review):

Summary:

The authors extend their earlier findings with bacterial adenylyl cyclases to mammalian enzymes. They show that certain aliphatic lipids activate adenylyl cyclases in the absence of stimulatory G proteins and that lipids can modulate activation by G proteins. Adding lipids to cells expressing specific isoforms of adenylyl cyclases could regulate cAMP production, suggesting that adenylyl cyclases could serve as 'receptors'.

Strengths:

This is the first report of lipids regulating mammalian adenylyl cyclases directly. The evidence is based on biochemical assays with purified proteins, or in cells expressing specific isoforms of adenylyl cyclases.

Weaknesses:

It is not clear if the concentrations of lipids used in assays are physiologically relevant. Nor is there evidence to show that the specific lipids that activate or inhibit adenylyl cyclases are present at the concentrations required in cell membranes. Nor is there any evidence to indicate that this method of regulation is seen in cells under relevant stimuli.

Although this question is not the subject of this manuscript, we will address this question in more detail in the discussion of the revision.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Weaknesses:

(1) At the beginning of the results section, the authors say "We have expected lipids as ligands". It is not quite clear why these could not have been other substances. It is because they were expected to bind in the lipophilic membrane anchors? Various lipophilic and hydrophilic ligands are known for GPCR which also have transmembrane domains. Maybe 1-2 additional sentences could be helpful here.

Will be done as suggested.

(2) In stably transfected HEK cells expressing mAC3 or mAC5, they have used only one dose of isoproterenol (2.5 uM) for submaximal AC activation. The reference 28 provided here (PMID: 33208818) did not specifically look at Iso and endogenous beta2 adrenergic receptors expressed in HEK cells. As far as I remember from the old pharmacological literature, this concentration is indeed submaximal in receptor binding assays but regarding AC activity and cAMP generation (which happen after signal amplification with a so-called receptor reserve), lower Iso amounts would be submaximal. When we measure cAMP, these are rather 10 to 100 nM but no more than 1 uM at which concentration response dependencies usually saturate. Have the authors tried lower Iso concentrations to prestimulate intracellular cAMP formation? I am asking this because, with lower Iso prestimulation, the subsequent stimulatory effects of AC ligands could be even greater.

The best way to address this issue is to establish a concentration-response curve for Iso-stimulated cAMP formation using the permanently transfected cells. We note that in the past isoproterenol concentrations used in biochemical or electrophysiological experiments differed substantially.

(3) The authors refer to HEK cell models as "in vivo". I agree that these are intact cells and an important model to start with. It would be very nice to see the effects of the new ligands in other physiologically relevant types of cells, and how they modulate cAMP production under even more physiological conditions. Probably, this is a topic for follow-up studies.

The last sentence is correct.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The authors have achieved their aims to a very high degree, their results do nicely support their conclusions. There is only one point (various classical GPCR concentrations, please see above) that would be beneficial to address.

Without any doubt, this is a groundbreaking study that will have profound implications in the field for the next years/decades. Since it is now clear that mammalian adenylyl cyclases are receptors for aliphatic fatty acids and anandamide, this will change our view on the whole signaling pathway and initiate many new studies looking at the biological function and pathophysiological implications of this mechanism. The manuscript is outstanding.