α1 adrenergic receptor - PKC - Pyk2 - Src signaling boosts L-type Ca2+ channel Cav1.2 activity and long-term potentiation in rodents

The cellular mechanisms mediating norepinephrine functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq- coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required PKC-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by norepinephrine. Indeed, a form of hippocampal LTP in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR - Pyk2 - Src signaling regulates synaptic strength.


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Gs, AC, and PKA are all associated with the L-type Ca 2+ channel (LTCC) Cav1.2 for efficient 36 signaling in neurons (7-12) and heart (13). The formation of this signaling complex identifies 37 Cav1.2 as a major effector of signaling by NE. We now find that Cav1.2 is also a major effector 38 for signaling via the 1AR, which has a higher affinity for NE than ARs (14, 15). Importantly a 39 large body of evidence implicates the 1AR in NE's role in attention and vigilance (16-22).   i.e., the metabotropic mGluR1/5 receptors with dihydroxyphenylglycine (DHPG) and muscarinic 149 receptors with muscarine, did not significantly increase LTCC activity (Fig. S1).          (Table   230   S1).

(D)
Pulldown assay of Pyk2 binding to 11.2 fragments. GST fusion proteins of the N-terminus, 232 the loops between domains I and II, II and III, III and IV, the whole C-terminus, and three 233 different overlapping fragments covering the C-terminus were expressed in E. coli, immobilized 234 on glutathione Sepharose, washed and incubated with purified His-tagged Pyk2. Comparable 235 amounts of fusion proteins were present (data not shown but see (11,69,97,139) Fig 8A). This LTPLTCC was completely blocked by the LTCC inhibitor nimodipine and 463 the 1AR antagonist prazosin (both P≤0.001, Fig 8A). Thus, this elevated potentiation strictly 464 depends on both the activity of LTCCs and signaling through 1ARs. Importantly, this LTPLTCC is 465 also blocked by the Pyk2 inhibitor PF-719 and the Src inhibitor PP2 (both P≤0.001, Fig 8B).

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These data indicate that robust LTPLTCC in 13-20 week old mice requires Pyk2 and Src activity 467 downstream of engaging 1AR to boost LTCC activation to sufficient levels.

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Firstly, we found that the 1AR-selective agonist PHE strongly increased LTCC activity in 495 neurons (Fig. 1). This increase was blocked by the 1AR-selective antagonist prazosin 496 confirming that the PHE effect was mediated by the 1AR. Stimulation of two other major 497 classes of Gq-coupled receptors in neurons, mGluR1/5 and muscarinic M1/3/5 receptors, did not 498 affect LTCC activity at the cell soma (Fig. S1). Accordingly, the 1AR augments LTCC activity 499 through stimulation of a specific Gqmediated signaling pathway rather than through general 500 activation of Gq. Thus, activity of these channels is precisely and judiciously regulated by 1AR 501 signaling. More generally, these observations indicate that certain Gq signals can selectively 502 target specific effector proteins. Defining what restricts the receptor type that can regulate to 503 LTCCs will be an interesting avenue of future investigation.

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Secondly, we identified a complex PKC/Pyk2/Src cascade that mediates regulation of Cav1.2 by 505 the 1AR. Clear evidence for this signaling pathway is provided by the inhibition of PHE-induced 506 upregulation of LTCC activity by inhibitors of PKC, Pyk2, and Src (Fig. 2), which is further 507 supported by the finding that direct stimulation of PKC also upregulates LTCC activity via Pyk2 508 and Src (Fig. 3). The role of the PKC/Pyk2/Src pathway in regulating Cav1.2 is also substantiated by the association of Pyk2 in addition to Src and PKC with Cav1.2 (Fig. 4) and 510 inhibition of PKC-induced tyrosine phosphorylation of Cav1.2 by Pyk2 and Src inhibitors (Fig. 6) 511 and Pyk2 and Src knockdown (Fig. 7). Multiple shRNAs specifically targeting both Pyk2 and Src 512 efficiently prevented the PKC-mediated increase in 11.2 tyrosine phosphorylation. These 513 observations not only confirm that Pyk2 mediates the Cav1.2 regulation downstream of PKC but 514 also indicates that Src itself is in this context the relevant member of the Src kinase family. 515 516 Thirdly, we found that Pyk2 is firmly associated with Cav1.2 under basal conditions as reflected 517 by their co-IP (Fig. 4). This association places Pyk2 into a complex that also contains its 518 immediate upstream activator and downstream effector, i.e., PKC and Src. PKC can directly 519 bind to the distal C-terminal region of 11.2, which also contain S1928 (66). Given that S1928 is 520 a phosphorylation site for PKC (66), it is conceivable that PKC binding to this region reflects a 521 temporary kinase -substrate interaction rather than a more permanent association of PKC with 522 Cav1.2, although an interaction with a phosphorylation site does not rule out that PKC can stably 523 bind to another region in the C-terminus of 11.2. In addition, the A kinase anchor protein 524 AKAP150, which is a major interaction partner for Cav1.2 (10, 79, 97), binds not only PKA but 525 also PKC (98) and constitutes another potentially constitutive link between PKC and Cav1.2 526 (67). Furthermore, like Pyk2, Src co-precipitates with Cav1.2 (Fig. 4)    In rat brain neurons LTCC activity is increased by Src via phosphorylation of 11.2 on Y2122 557 (62, 65). However, this phosphorylation site is not conserved even within rodents. It is 558 equivalent to position 2150 in rabbit cardiac 11.2, which is a Cys not Tyr residue (99). Accordingly, other Tyr residues must serve as phosphorylation sites. It will be an interesting 560 challenge for future work to identify the exact phosphorylation site and then test its functional 561 relevance.

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Remarkably, the Src inhibitor PP2 also completely blocked PKC-induced autophosphorylation of 564 Pyk2 on Y402 (Fig. 5J,K). This finding indicates a close interdependence between Pyk2 and Src 565 activation by PKC in PC12 cells. It is consistent with earlier results indicating that Pyk2

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Prolonged theta tetanus LTP (PTT-LTP), which is induced by a 3-minute-long 5 Hz tetanus, also 582 depends on upregulation of Cav1.2 activity (28, 29, 92, 93, 115). In PTT-LTP this upregulation is 583 accomplished by 2AR -Gs -adenylyl cyclase/cAMP -PKA signaling and the ensuing 585 96). Whether signaling by NE through 1AR and 2AR can act in parallel and is additive will be 586 an interesting question for future studies. However, we already know that at least for classic 587 PTT-LTP 2AR signaling is sufficient and does not require engagement of 1AR signaling (96).

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Because regulation of Cav1.2 by 2AR signaling is highly localized (7, 11), it is conceivable that 589 1AR signaling might engage a subpopulation of Cav1.2 channels whose spatial distribution