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Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling

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ABSTRACT

GPCRs can activate different programs of gene expression from the plasma membrane and the endosome. Bowman et al. show that signaling by endosomal β-2 adrenergic receptors occurs at the microdomains that GPCRs use for sequence-dependent recycling.

No MeSH data available.


A phosphomimetic B2AR mutant is restricted to ASRT domains independent of PKA and activates Gαs in ASRT domains. (A) Example images of B2AR and SS>DD ASRT (arrowheads) and non-ASRT (arrows) tubules after iso and before and after KT. Bar, 2 µm. (B) Percent ASRT tubules (marked by coronin) and non-ASRT tubules for B2AR and SS>DD with and without KT. KT does not cause SS>DD to enter non-ASRT tubules. n = 43 (B2AR − KT), 26 (B2AR + KT), 64 (SS>DD − KT), and 36 (SS>DD + KT) cells. n.s., not significant. (C) Example images of FLAG-B2AR and SS>DD with cortactin and Nb37 after iso. Nb37 is recruited to SS>DD ASRT tubules (arrowheads). Bar, 2 µm. (D) Percent tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither marker. n = 42 (B2AR) and 47 (SS>DD) cells. Error bars in all graphs are SEM across cells.
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fig3: A phosphomimetic B2AR mutant is restricted to ASRT domains independent of PKA and activates Gαs in ASRT domains. (A) Example images of B2AR and SS>DD ASRT (arrowheads) and non-ASRT (arrows) tubules after iso and before and after KT. Bar, 2 µm. (B) Percent ASRT tubules (marked by coronin) and non-ASRT tubules for B2AR and SS>DD with and without KT. KT does not cause SS>DD to enter non-ASRT tubules. n = 43 (B2AR − KT), 26 (B2AR + KT), 64 (SS>DD − KT), and 36 (SS>DD + KT) cells. n.s., not significant. (C) Example images of FLAG-B2AR and SS>DD with cortactin and Nb37 after iso. Nb37 is recruited to SS>DD ASRT tubules (arrowheads). Bar, 2 µm. (D) Percent tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither marker. n = 42 (B2AR) and 47 (SS>DD) cells. Error bars in all graphs are SEM across cells.

Mentions: To confirm that phosphorylation of B2AR at S345 and S346 confines Gαs activation to ASRT domains, we generated a phosphomimetic version of B2AR, mutating both S345 and S346 to aspartic acid (SS>DD). SS>DD localized exclusively to ASRT domains (Fig. 3 A). Importantly, unlike wild-type B2AR, PKA inhibition did not redistribute SS>DD into constitutive tubules (Fig. 3, A and B), suggesting that phosphorylation of S345 and S346 directly regulates the sorting of B2AR into ASRT domains. Nb37 was recruited only to B2AR and to SS>DD domains overlapping with cortactin (Fig. 3, C and D; and Fig. S2 B), together suggesting that B2ARs, when phosphorylated at S345 and S346, are sorted into ASRT domains, where they activate Gαs. ASRT domains therefore might function as specific scaffolds for recruiting G proteins to the endosome.


Distinct G protein – coupled receptor recycling pathways allow spatial control of downstream G protein signaling
A phosphomimetic B2AR mutant is restricted to ASRT domains independent of PKA and activates Gαs in ASRT domains. (A) Example images of B2AR and SS>DD ASRT (arrowheads) and non-ASRT (arrows) tubules after iso and before and after KT. Bar, 2 µm. (B) Percent ASRT tubules (marked by coronin) and non-ASRT tubules for B2AR and SS>DD with and without KT. KT does not cause SS>DD to enter non-ASRT tubules. n = 43 (B2AR − KT), 26 (B2AR + KT), 64 (SS>DD − KT), and 36 (SS>DD + KT) cells. n.s., not significant. (C) Example images of FLAG-B2AR and SS>DD with cortactin and Nb37 after iso. Nb37 is recruited to SS>DD ASRT tubules (arrowheads). Bar, 2 µm. (D) Percent tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither marker. n = 42 (B2AR) and 47 (SS>DD) cells. Error bars in all graphs are SEM across cells.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037407&req=5

fig3: A phosphomimetic B2AR mutant is restricted to ASRT domains independent of PKA and activates Gαs in ASRT domains. (A) Example images of B2AR and SS>DD ASRT (arrowheads) and non-ASRT (arrows) tubules after iso and before and after KT. Bar, 2 µm. (B) Percent ASRT tubules (marked by coronin) and non-ASRT tubules for B2AR and SS>DD with and without KT. KT does not cause SS>DD to enter non-ASRT tubules. n = 43 (B2AR − KT), 26 (B2AR + KT), 64 (SS>DD − KT), and 36 (SS>DD + KT) cells. n.s., not significant. (C) Example images of FLAG-B2AR and SS>DD with cortactin and Nb37 after iso. Nb37 is recruited to SS>DD ASRT tubules (arrowheads). Bar, 2 µm. (D) Percent tubules per cell that contain cortactin and Nb37, cortactin or Nb37 only, or neither marker. n = 42 (B2AR) and 47 (SS>DD) cells. Error bars in all graphs are SEM across cells.
Mentions: To confirm that phosphorylation of B2AR at S345 and S346 confines Gαs activation to ASRT domains, we generated a phosphomimetic version of B2AR, mutating both S345 and S346 to aspartic acid (SS>DD). SS>DD localized exclusively to ASRT domains (Fig. 3 A). Importantly, unlike wild-type B2AR, PKA inhibition did not redistribute SS>DD into constitutive tubules (Fig. 3, A and B), suggesting that phosphorylation of S345 and S346 directly regulates the sorting of B2AR into ASRT domains. Nb37 was recruited only to B2AR and to SS>DD domains overlapping with cortactin (Fig. 3, C and D; and Fig. S2 B), together suggesting that B2ARs, when phosphorylated at S345 and S346, are sorted into ASRT domains, where they activate Gαs. ASRT domains therefore might function as specific scaffolds for recruiting G proteins to the endosome.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

GPCRs can activate different programs of gene expression from the plasma membrane and the endosome. Bowman et al. show that signaling by endosomal β-2 adrenergic receptors occurs at the microdomains that GPCRs use for sequence-dependent recycling.

No MeSH data available.