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J. Biol. Chem., Vol. 280, Issue 25, 23771-23777, June 24, 2005

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Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone

EVIDENCE FOR BINDING TO BOTH THE N-TERMINAL EXTRACELLULAR DOMAIN AND EXTRACELLULAR LOOP REGION^*

Jean-René Barbier, Thomas J. Gardella, Thomas Dean, Susanne MacLean, Zhanna Potetinova, James F. Whitfield, and Gordon E. Willick¶

From the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6 Canada and Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

We have used backbone N-methylations of parathyroid hormone (PTH) to study the role of these NH groups in the C-terminal amphiphilic -helix of PTH (1–31) in binding to and activating the PTH receptor (P1R). The circular dichroism (CD) spectra indicated the structure of the C-terminal -helix was locally disrupted around the methylation site. The CD spectra differences were explained by assuming a helix disruption for four residues on each side of the site of methylation and taking into account the known dependence of CD on the length of an -helix. Binding and adenylyl cyclase-stimulating data showed that outside of the -helix, methylation of residues Asp³⁰ and Val³¹ had little effect on structure or activities. Within the -helix, disruption of the structure was associated with increased loss of activity, but for specific residues Val²¹, Leu²⁴, Arg²⁵, and Leu²⁸ there was a dramatic loss of activities, thus suggesting a more direct role of these NH groups in correct P1R binding and activation. Activity analyses with P1R-delNT, a mutant with its long N-terminal region deleted, gave a different pattern of effects and implicated Ser¹⁷, Trp²³, and Lys²⁶ as important for its PTH activation. These two groups of residues are located on opposite sides of the helix. These results are compatible with the C-terminal helix binding to both the N-terminal segment and also to the looped-out extracellular region. These data thus provide direct evidence for important roles of the C-terminal domain of PTH in determining high affinity binding and activation of the P1R receptor.

Received for publication, January 24, 2005 , and in revised form, April 8, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I.

^¶ To whom correspondence should be addressed: Institute for Biological Sciences, National Research Council, Ottawa, ON, K1A 0R6, Canada. Tel.: 613-990-0852; Fax: 613-952-9092; E-mail: gordon.willick{at}nrc.ca.

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