Receptor recruitment: a mechanism for interactions between G protein-coupled receptors

Abstract

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization.

Figure 1

Effect of ANP on localization of D1 DA receptors. Confocal recordings of D1 DA receptors were taken from midsections of LLCPK cells. The color-scale used is a pseudo-color-scale, with blue indicating low fluorescence signal and red–white indicating high fluorescence signal. The images shown are representative for at least three separate experiments. (a) The D1 DA receptor signal is found throughout untreated cells. (b) In samples in which the primary antibody had been preabsorbed with the peptide against which the antibody was raised, there was no detectable fluorescence signal. (c–j) Cells were treated with drugs for 1 min. Treatment with ANP at 10⁻⁹ M (c) or 10⁻⁶ M (d) resulted in a distinct signal in the region of the plasma membrane. This effect was sustained for at least 5 min (not shown). (e) cGMP (10⁻⁴ M) also resulted in an enhanced signal in the region of the plasma membrane. (f) In cells treated with the D1 DA receptor antagonist SCH 23390 (10⁻⁶ M), the effect of ANP (10⁻⁶ M) was abolished. (g) Dideoxyadenosine (DDA; 10⁻⁴ M) also abolished the effect of ANP (10⁻⁶ M) on D1 DA receptor localization. DA (10⁻¹⁰ M; h) and ANP (10⁻¹¹ M; i) had no effect on receptor localization when added alone but did, in combination, enhance the D1 DA receptor signal in the region of the plasma membrane (j). SCH 23390 or DDA alone had no effect on D1 DA receptor localization (data not shown).

Figure 2

Localization of the D1 DA receptor by subcellular fractionation. Rat renal outer cortical tissue slices were incubated with or without ANP (10⁻⁶ M) for 15 min, subjected to subcellular fractionation, and immunoblotted for D1 DA receptor. The amount of the integral membrane protein Na⁺,K⁺-ATPase, used as a marker for the plasma membrane, was highest in fraction 1 and not detectable in fractions 3–10 in vehicle-treated tissue. The plasma membrane fraction was therefore identified as fraction 1. In vehicle-treated tissue, the D1 DA receptor subtype was located mainly in fraction 7 (reference fraction).

Figure 3

Effect of ANP and DA on the activity of Na⁺,K⁺-ATPase in single permeable rat proximal tubule segments. ANP (10⁻⁶ M) significantly decreased Na⁺,K⁺-ATPase activity. (a) This effect was abolished by the D1 DA receptor antagonist SCH 23390 (10⁻⁶ M). (b) In tubules incubated with both ANP (10⁻⁸ M) and DA (5 × 10⁻⁸ M), Na⁺,K⁺-ATPase activity was decreased by 48 ± 10%. In each experiment, 5–8 tubules from the same rat were studied in each protocol (vehicle or drug incubation). Average values from these tubules were used as one data point. Data in a and b are means ± SEM for data from three rats. ∗, P < 0.05 vs. control; NS, not significant vs. control. Statistical analysis was performed with paired and unpaired Student’s t test.

Figure 4

Effect of NPY on localization of α_1A-adrenergic receptors. Confocal micrographs show the localization of the α_1A-adrenergic receptor in LLCPK cells. (a) In untreated cells, the α_1A-adrenergic receptor immunofluorescence signal was found throughout the cell. (b) In sections in which the primary antibody had been preabsorbed with the peptide against which the antibody was raised, there was no detectable fluorescence signal. (c) After treatment with the α_1A-adrenergic receptor agonist phenylephrine (10⁻⁶ M) for 10 s, a distinct signal was observed in the region of the plasma membrane. (d) After 60 s, α_1A-adrenergic receptor immunofluorescence signal was again observed throughout the cell. (e) The relocalization of the α_1A-adrenergic receptors at 10 s was not observed in cells pretreated with the α_1A-adrenergic receptor antagonist prazosin (10⁻⁶ M). Incubation with phenylephrine (10⁻¹⁰ M) alone (f) or NPY (10⁻¹⁰ M) alone (g) for 10 s had no effect on receptor localization, but when applied in combination (h), there was a marked and rapid increase of the α_1A-adrenergic receptor signal in the region of the plasma membrane. (i) This effect was sustained for at least 5 min. (j) Incubation with NPY (10⁻⁸ M) alone for 10 s caused a rapid relocalization of the α_1A-adrenergic receptors to the region of the plasma membrane. (k) This effect was sustained for at least 5 min. (l) The effect of NPY (10⁻⁸ M) at 10 s was not seen in cells pretreated with the α₁-adrenergic receptor antagonist prazosin (10⁻⁶ M).

Figure 5

Localization of the α_1A-adrenergic receptor by subcellular fractionation. Rat renal outer cortical tissue slices were incubated with or without NPY (10⁻⁶ M) plus phenylephrine (10⁻⁶ M) for 15 min, subjected to subcellular fractionation, and immunoblotted for α_1A-adrenergic receptors. For experimental details, see legend to Fig. 2.

Figure 6

Peptide-induced accumulation of D1 DA and α_1A-adrenergic receptors at the plasma membrane is attributable to increased receptor recruitment. Control cells immunolabeled for D1 DA receptor (a) or α_1A-adrenergic receptor (b) did not show an increased signal in the region of the plasma membrane after pretreatment with the endocytosis inhibitor PAO (10⁻⁵ M) for 3 min. (c and d) In cells treated with phenylephrine (10⁻⁶ M) for 10 s, an increased α_1A-adrenergic receptor signal was found in the region of the plasma membrane. (e) After a 3-min treatment with phenylephrine, the α_1A-adrenergic receptor signal was observed throughout the cell. (f) This internalization of α_1A-adrenergic receptors was abolished after PAO treatment.