Figure 4.

(A) Internalization of HA-β1-AR in HEK293 cells is potentiated on overexpression of SH3p4. HEK293 cells were transiently transfected with 0.5–1 μg of pCDNA1 HA-β1-AR or HA-β2-AR to obtain a receptor expression level of 500 fmol/mg protein in the presence or absence of pcDNA3 Flag-SH3p4. Cells were stimulated with 10 μM isoproterenol for 30 min. Results are presented as the percentage loss of cell surface receptor. For HA-β1-AR, n = 9, P < 0.01(∗∗) for comparison of HA-β1-AR internalization in the presence versus absence of SH3p4 according to Student’s t test; for HA-β2-AR, n = 5. (B) Overexpression of SH3p4 in HEK293 cells attenuates HA-β1-AR stimulated adenylyl cyclase activity. Stable HEK293 cells, overexpressing pcDNA3 HA-SH3p4, pcDNA3 HA-SH3p4 ΔSH3, or pcDNA3 empty vector, were transfected with pCDNA1 HA-β1-AR to achieve a receptor concentration of ≈500 fmol/mg protein. HA-β1-AR was stimulated for 10 min with the indicated concentrations of dobutamine. Whole cell cAMP accumulation was measured as the percentage conversion of [³H]adenine to [³H]cAMP. cAMP accumulation on 50 μM forskolin stimulation was used to normalize dobutamine-induced cAMP production. The cyclase activity observed in the presence of HA-β1-AR overexpression in the pcDNA3-stable HEK293 cells at 10 μM dobutamine is set at 100. The other data points are shown as percentage of this maximal cyclase activity. The means ± SE from four independent experiments are shown.