Determinants Present in the Receptor Carboxy Tail Are Responsible for Differences in Subtype-Specific Coupling of beta-Adrenergic Receptors to Phosphoinositide 3-Kinase

Abstract

An agonist-occupied beta(2)-adrenergic receptor (beta(2)-AR) recruits G protein receptor kinase-2 (GRK2) which is recruited to the membrane. Thus, the physical proximity of activated beta(2)-AR and PI-3K allows the activation of the latter. In contrast, it has been observed that the beta(1)-AR is unable to activate the PI-3K/Akt pathway. We hypothesized that the difference might be due to molecular determinants present in the carboxy termini of the two beta-AR subtypes. Using transiently transfected HEK 293 cells expressing either beta(1)- or beta(2)-AR, we also observed that in presence of an agonist, beta(2)-AR, but not beta(1)-AR, is able to activate the PI-3K/Akt pathway. Switching the seventh transmembrane domain and the carboxy tail between the two receptors reverses this phenotype; that is, beta(1) x beta(2)-AR can activate the PI-3K/Akt pathway whereas beta(2) x beta(1)-AR cannot. Pretreatment with pertussis toxin abolished the activation of PI-3K by beta(2)- or beta(1) x beta(2)-AR stimulation. Ligand-mediated internalization of the beta(2)-AR induced by a 15-minute stimulation with agonist was abolished in the presence of a dominant negative of PI-3K or following pertussis toxin pretreatment. These results indicate that the subtype-specific differences in the coupling to PI-3K/Akt pathway are due to molecular determinants present in the carboxy tail of the receptor and further that beta(2)-AR activates PI-3K via a pertussis toxin-sensitive mechanism.

Figure 1

PI-3K activity with β-AR stimulation. HEK 293 cells were transfected with β ₁-AR or β ₂-AR. Forty-eight hours after transfection, cells were stimulated with 1 μM isoproterenol for the indicated times. PI-3K activity was determined by the level of [³²P]-PI produced by the stimulation of β ₁-AR (□) or β ₂-AR (■) expressing cells. Top panel is a representative autoradiograph of TLC separation (n = 4−5). Data from these experiments is quantitated in the bottom panel as described in Section 2 . *P < .05 versus 0 minute.

Figure 2

Akt activation following β-AR stimulation. HEK 293 cells were transfected with β ₁-AR or β ₂-AR. Forty-eight hours after the transfection, cells were stimulated with 1 μM isoproterenol for the indicated times. Akt activation status was determined by immunoblot for the phosphorylated form of Akt compared to the total amount of Akt after stimulation of β ₁-AR (□) or β ₂-AR (■) expressing cells. Top panel is a representative immunoblot of the experiments (n = 4−5). Data from these experiments is quantitated in the bottom panel as described in Section 2 . *P < .05 versus 0 minute.

Figure 3

PI-3K Partitioning of PI-3K to the membrane. Forty-eight hours after transfection, cells were stimulated with 1 μM isoproterenol for the indicated times. Membranes were isolated, solubilized, and approximately 100 μg of membrane proteins were separated on a 10% SDS-PAGE gel. Membrane-associated PI-3K levels increased with cells expressing β ₁-AR (□) or β ₂-AR (■). Top panel is a representative immunoblot of the experiments (n = 3−4). Data from these experiments is quantitated in the bottom panel as described in Section 2 . *P < .05 versus 0 minute.

Figure 4

GRK2 carboxy-terminal domain reduced PI-3K recruitment to membrane fractions in response to β-AR stimulation. HEK 293 cells were cotransfected with ct-GRK-2 and β-AR subtypes. Forty-eight hours after the transfection, cells were stimulated with 1 μM isoproterenol for 5 minutes. PI3-kinase recruitment to particulate fractions decreased with cells expressing either β ₁-AR (□) or β ₂-AR (■) in presence of cotransfected ct-GRK2. Top panel is a representative immunoblot of the experiments (n = 4–6). Data from these experiments is quantitated in the bottom panel as described in Section 2. *P < .05 versus 0 minute.

Figure 5

PI-3K activity with chimeric β-AR stimulation. HEK 293 cells were transfected with β ₁ × β ₂-AR or β ₂ × β ₁-AR. Forty-eight hours after transfection, cells were stimulated with 1 μM isoproterenol for the indicated times. PI-3K activity was determined by the level of [³² P]-PI produced by the stimulation of β ₁ × β ₂-AR (□) or β ₂ × β ₁-AR (■) expressing cells. Top panel is a representative autoradiograph of the TLC separation (n = 4−5). Data from these experiments is quantitated in the bottom panel as described in Section 2. *P < .05 versus 0 minute.

Figure 6

Akt activity with chimeric β-AR stimulation. HEK 293 cells were transfected with β ₁ × β ₂-AR or β ₂ × β ₁-AR. Forty-eight hours after the transfection, cells were stimulated with 1 μM isoproterenol for the indicated times. Akt activity was determined by measuring levels of phosphorylated Akt compared with total Akt produced by the stimulation of β ₁ × β ₂-AR (□) or β ₂ × β ₁-AR (■) expressing cells. Upper panel is a representative immunoblot of the experiments (n = 6−7). Data from these experiments is quantitated in the bottom panel as described in Section 2. *P < .05 versus 0 minute.

Figure 7

Pertussis toxin effects on β-AR-mediated PI-3K activation. Twenty-six hours after transfection, cells were incubated with 0.1 μg/mL pertussis toxin for 18 hours. Cells were stimulated for 5 minutes with 1 μM isoproterenol prior to processing for TLC as described in Figure 1 and Section 2. PI-3K activity was determined by the level of [³² P]-PI produced by the stimulation of β ₁-AR (a), β ₂-AR (b), β ₂ × β ₁-AR (c), and β ₁ × β ₂-AR expressing cells. (n = 3−4; *P < .05 versus basal and PTX-Iso conditions).

Figure 8

p85ΔPI-3K reduced agonist-induced β₂-AR sequestration. HEK 293 cells were transfected with p85ΔPI-3K and the two β-AR subtypes. Cells were stimulated with 1 μM isoproterenol for 15 minutes (b) and (f). Stimulation of β ₂-AR-transfected cells induced a cluster distribution which is characteristic of receptor internalization (f). This sequestration was abolished in presence of either cotransfected p85ΔPI-3K (g) or PTX pretreatment (h). No significant β ₁-AR sequestration was observed under the different conditions used in these experiments (a)–(d). Representative images of several experiments (n = 8–15) are shown.