Differential activation of mitogen-activated protein kinase pathways in PC12 cells by closely related α1-adrenergic receptor subtypes

Coupling of the three known alpha(1)-adrenergic receptor (alpha(1)-AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing alpha(1A)-, alpha(1B)-, and alpha(1D)-ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2+ level in these cells in a manner dependent on receptor density. However, alpha(1A)-ARs activated these second messenger responses more effectively than alpha(1B)-ARs, whereas alpha(1D)-ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three alpha(1)-AR subtypes, although alpha(1A)- and alpha(1B)-ARs caused larger ERK activation than did alpha(1D)-ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in alpha(1A)- and alpha(1B)- but not alpha(1D)-transfected cells and activated c-Jun NH2-terminal kinase (JNK) only in alpha(1A)-transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in alpha(1A)-transfected PC12 cells. NE caused neurite outgrowth only in alpha(1A)-expressing PC12 cells, but not in alpha(1B)- or alpha(1D)-transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that alpha(1A)-ARs activate all three MAPK pathways, alpha(1B)-ARs activate ERKs and p38 but not JNKs, and alpha(1D)-ARs only activate ERKs. Only the alpha(1A)-AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.