Hypoxia stabilizes the β₂AR by inhibiting its ubiquitylation. (A) FLAG-tagged β₂AR was isolated with anti-FLAG beads from β₂AR-293 cells cultured under 21 or 1% O₂ with or without 10 μM isoproterenol (ISO) for 18 hours, and Western blotted with anti-β₂AR antibody. Actin was used as the loading control. n = 3 (n = the number of independent experiments, here and elsewhere), *P< 0.01 versus 21%, **P< 0.05 versus 21% + ISO (Student's t test). (B) HUVECs were cultured under 21 or 1% O₂ with or without 10 μM ISO for 18 hours. Cell membranes were collected and β₂AR density was measured by radioligand binding (n = 2; each bar represents the result of an individual experiment). (C) β₂AR-293 cells were metabolically labeled with ³⁵S[Met] and ³⁵S[Cys] for 12 hours followed by a chase period of the indicated durations carried out at 21 or 1% O₂. FLAG-β₂AR was immunoprecipitated from the cell lysates and signals were detected by SDS-PAGE followed by autoradiography. n = 3, *P < 0.05 versus 1% by analysis of variance (ANOVA). (D) β₂AR-293 cells were treated with MG132 (10 μM), lactacystin (20 μM), or chloroquine (20 μM) for 18 hours. Receptor was collected with anti-FLAG beads and Western blotted with anti-β₂AR. (E) β₂AR was collected with anti-FLAG beads from β₂AR-293 cells cultured at 21 or 1% O₂ with MG132 (10 μM) for 18 hours and Western blotted with anti-ubiquitin or anti-β₂AR. n = 3, *P < 0.01 versus 21% (Student's t test). In (A), (C), and (E), densitometric analysis (mean ± SEM) was performed.

The β₂AR is hydroxylated at proline residues P³⁸² and P³⁹⁵. (A and B) FLAG-pVHL and β₂AR [(A) and (B)] or β₂AR^P382A/P395A (B) were cotransfected into HEK293 cells; in (A), the leftmost lane shows the result of control transfection with pcDNA3 rather than pcDNA3-β₂AR. After 24 hours, cells were incubated for an additional 18 hours at either 21 or 1% O₂ in the presence of 10 μM MG132. Cell lysates were immunoprecipitated with anti-β₂AR and Western blotted with anti-FLAG (for pVHL) or anti-β₂ARs. (C) HEK293 cells stably expressing FLAG-β₂AR or FLAG-β₂AR^P382A/P395A were incubated at 21 or 1% O₂ or treated with 1 mM DMOG for 18 hours at 21% O₂. β₂AR or β₂AR^P382A/P395A was isolated with anti-FLAG beads and Western blotted with anti-β₂AR antibody. Actin was used as loading control. (D) FLAG-β₂AR purified from β₂AR-293 cells cultured at 21% O₂ was digested with trypsin and analyzed by LC-MS. Monoisotopic precursor ions (shown in red) at m/z = 1052.83 and 1058.16 correspond to [M+3H]⁺³ of the unhydroxylated and hydroxylated LLCEDLPGTEDFVGHQGTVPSDNIDSQGR peptides, respectively. (E) Tandem mass spectrum of the precursor ion at m/z = 1058.16. Large red “P” indicates a hydroxylated proline residue. The peak heights are the relative abundances of the corresponding fragment ions, with the annotation of the identified matched N terminus–containing ions (b ions) in blue and C terminus–containing ions (y ions) in magenta. For clarity, only the major identified peaks are labeled (a complete table of fragment ions for this spectrum is presented in fig. S3B).

EGLN3 serves as an endogenous hydroxylase for the β₂AR. (A) β₂AR-293 cells (expressing FLAG-β₂AR) were transfected with EGLN1 or EGLN3 siRNA for 48 hours, and cell lysates were Western blotted with anti-β₂AR, anti-EGLN1, anti-EGLN3, and anti-actin. Densitometric analysis (mean ± SEM) was performed. n = 3, *P < 0.01 versus siRNA-scramble (control). (B) GST-β₂AR(330–413) immobilized on GSH-Sepharose was incubated with [³⁵S]pVHL (60 min, 4°C) either directly or after three 60-min incubations with fresh HEK293 cell lysate. The beads were eluted and the eluants were analyzed by SDS-PAGE and autoradiography. The bottom panel shows the inputs of GST-β₂AR(330–413). (C) Immobilized GST-β₂AR(330–413) was incubated with lysates from HEK293 cells transfected with siRNA to EGLN3 or control (scrambled) siRNA and digested with trypsin (37°C, 16 hours). Digests were analyzed by MALDI-TOF. Peaks at m/z = 3099.414 or 3099.375 and 3115.402 correspond to unhydroxylated and hydroxylated LLCEDLPGTEDFVGHQGTVPSDNIDSQGR peptides, respectively. (D) βARs densities on membranes derived from hearts of wild-type (wt) and EGLN3 knockout mice were measured by radioligand binding. n = 7, *P < 0.02 versus wild type and **P < 0.01 versus wild type (Student's t test).

The pVHL complex associates with the β₂AR and serves as an E3 ubiquitin ligase. (A) β₂AR-293 cells were transiently transfected with pVHL-GFP, fixed, and stained with anti– FLAG M2 (β₂AR) and TRITC-conjugated secondary antibody (red). (B) FLAG-pVHL and β₂AR or pcDNA3 were cotransfected into HEK293 cells. ISO stimulation was for 15 min. Cell lysates were immunoprecipitated (IP) with anti-β₂AR and Western blotted (IB) with anti-β₂AR, anti-FLAG (for pVHL), anti–cullin-2, or anti–elongin-C as indicated. (C and D) In vitro ubiquitylation assays were performed for recombinant β₂AR (150-kD dimer in C is reflective of high recombinant protein concentration) in the presence or absence of pVHL complex or E1 ubiquitin-conjugating enzyme. Samples were Western blotted with anti-β₂AR (C) or anti-ubiquitin (D). Bottom panels show the pVHL input. (E) HUVECs were electrotransfected with scrambled siRNA, siRNAs for human VHL, or plasmid expressing HIF-1α^P402A/P564G for 72 hours. Cell membranes were collected and β₂AR density was measured by radioligand binding. n = 3, *P < 0.01 versus scrambled siRNA (Student's t test). (F) β₂AR was transfected into 786-O or 786-O–pVHL cells; after 24 hours, cells were incubated for an additional 18 hours at 21 or 1% O₂. Proteins were Western blotted with specific antibodies as indicated. The illustrated pVHL-dependent effects of PO₂ are representative of three experiments, but cotransfection with pVHL had variable effects on the overall abundance of transfected β₂AR in 786-O cells.

EGLN3 interacts specifically with the β₂AR. (A) HEK293 cells were cotransfected with FLAG-EGLN1, 2, or 3 and with β₂AR or pcDNA3 control. Cell lysates were immunoprecipitated with anti-β₂AR and Western blotted with anti-β₂AR or anti-FLAG (for EGLNs). (B) β₂AR-293 cells were transfected with truncated FLAG-EGLN3 constructs. Cell lysates were immunoprecipitated with anti-β₂AR and Western blotted with anti-FLAG or anti-β₂AR. (C) GST or truncated GST-EGLN3(89–104) fusion protein (5 μg) were incubated with an equal amount of β₂AR before GST pulldown. Pulldowns and inputs were separated by SDS-PAGE and Western blotted with anti-β₂AR or stained with Coomassie blue (for GST or the fusion protein). (D) GST or GST-β₂AR(330–413) (GST-β₂AR-c-tail) (5 μg) were used to pull down EGLN3. Pulldowns were Western blotted with anti-EGLN3 antibody. (E) 100 nM recombinant EGLN3 and 100 nM GST or GST-β₂AR-c-tail were incubated in the presence of different concentrations of FITC-labeled peptide derived from human EGLN3(89–104) or mutant peptide EGLN3(4R). GST or GST fusion were then pulled down and Western blotted with anti-EGLN3. (F) HEK293 cells were transfected with pcDNA3 or FLAG-β₂AR and treated with 30 μM EGLN3(89–104) peptide as indicated for 4 hours. Cell lysates were immunoprecipitated with anti-FLAG beads and Western blotted with anti-EGLN3 or anti-β₂AR as indicated. (G) β₂AR-293 cells were treated with different concentrations of EGLN3(89–104) and EGLN3(4R) mutant peptides or 1 mM DMOG as indicated for 18 hours. β₂ARs were collected by anti-FLAG beads and Western blotted with anti-β₂AR. Actin was the loading control. Densitometric analysis (mean ± SEM) was performed; n = 3, *P < 0.01 versus control, ⁺P < 0.05 versus control (Student's t test).